Mice That Produce Antigen-Binding Proteins With pH-Dependent Binding Characteristics

ABSTRACT

Genetically modified non-human animals are provided that comprise an immunoglobulin heavy chain locus comprising an unrearranged human heavy chain variable region nucleotide sequence comprising an addition of at least one histidine codon or a substitution of at least one endogenous non-histidine codon with a histidine codon. Compositions and methods for making the genetically modified non-human animals as described herein are provided. Non-human animals capable of expressing an antigen-binding protein characterized by pH-dependent antigen binding, enhanced recyclability and/or enhanced serum half-life are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/832,309, filed 15, Mar. 2013, entitled “Mice That ProduceAntigen-Binding Proteins With pH-Dependent Binding Characteristics,”which claims the benefit of priority to U.S. Provisional Application No.61/611,950, filed 16, Mar. 2012, U.S. Provisional Application No.61/613,352, filed Mar. 20, 2012, and U.S. Provisional Application No.61/736,930, filed 13, Dec. 2012, the entire contents of each of theapplications are incorporated herein by reference.

FIELD OF THE INVENTION

Genetically modified immunoglobulin loci of non-human animals comprisingan unrearranged human heavy chain variable region nucleotide sequence,wherein the unrearranged human heavy chain variable region nucleotidesequence comprises an addition of least one histidine codon or asubstitution of at least one non-histidine codon with a histidine codon.Non-human animals, including rodents, e.g., mice and rats, comprising intheir germline an unrearranged human immunoglobulin heavy chain variableregion nucleotide sequence, wherein the unrearranged humanimmunoglobulin heavy chain variable region nucleotide sequence comprisesan addition of least one histidine codon or a substitution of at leastone non-histidine codon with a histidine codon. Genetically engineerednon-human animals capable of expressing an antigen-binding protein thatis characterized by pH-dependent antigen binding, improvedrecyclability, and/or enhanced serum half-life.

BACKGROUND OF THE INVENTION

Drugs administered into the body, including therapeutic monoclonalantibodies, can be affected via various elimination mechanisms,including glomerular filtration (e.g., into urine), secretion (e.g.,into the bile), and catabolism by cells. While small molecules arecleared from the body via renal filtration, the majority of secretedantibodies (e.g., IgG, which are too big to be filtered throughglomeruli) are primarily removed from the body via cell-mediatedcatabolism, e.g., fluid-phase endocytosis (phagocytosis) orreceptor-mediated endocytosis. For example, soluble molecules withseveral repeated epitopes are bound by a plurality of circulatingantibodies, and the resulting large antigen-antibody complexes arephagocytosed rapidly into cells for degradation. On the other hand, cellsurface target receptors, which are bound by antibodies (i.e.,receptor-antibody complexes), undergo target-mediated endocytosis in adose-dependent manner, which leads to formation of endosomes destinedfor lysosomal degradation inside cells. In some cases, the endocytosedreceptor-antibody complexes bind neonatal Fc receptors (FcRn) inside theendosomes in a pH-dependent manner and are routed back to the cellsurface for release into plasma or interstitial fluids upon exposure toa neutral extracellular pH (e.g., pH 7.0-7.4).

There is a need in the art for systems, e.g., non-human animals, cells,and genomic loci that generate antigen-binding proteins with titratableresidues, e.g., genetically modified loci that rearrange immunoglobulingene segments to generate heavy chain variable domains that respond tochanges in pH, e.g., that donate or accept protons and, e.g., whosebinding characteristics differ according to protonation state.

There is also a need in the art for methods and compositions that canfurther increase recycling efficiency of endocytosed antigen-bindingproteins by promoting dissociation of antigen-binding proteins fromreceptor-antigen-binding protein complexes or by increasing the affinityof antigen-binding proteins toward FcRn in an acidic endosomalcompartment without compromising the specificity and affinity of theantigen-binding protein toward an antigen of interest.

SUMMARY OF THE INVENTION

Genetically modified immunoglobulin heavy chain loci in the germlinegenome of non-human animals are provided, wherein the immunoglobulinheavy chain loci comprise a genetically modified unrearranged heavychain variable region nucleotide sequence (e.g., one or more geneticallymodified human V_(H), D, and/or J_(H) gene segment), wherein theunrearranged heavy chain variable region nucleotide sequence comprisesan addition of at least one histidine codon or a substitution of atleast one endogenous non-histidine codon with a histidine codon. Invarious embodiments, the genetically modified unrearranged heavy chainvariable region nucleotide sequence comprises at least one histidinecodon in at least one reading frame that encodes an immunoglobulin heavychain variable domain. In various embodiments, the unrearranged heavychain variable region nucleotide sequence comprising the at least onehistidine codon is operably linked to a human or non-human heavy chainconstant region nucleotide sequence (e.g., a heavy chain constant regionnucleotide sequence that encodes an immunoglobulin isotype selected fromIgM, IgD, IgA, IgE, and IgG).

Non-human animals (mammals, e.g., rodents such as mice, rats, orhamsters) are provided that are genetically engineered to containimmunoglobulin heavy chain genomic loci in their germline genome,wherein the genomic loci comprise an unrearranged heavy chain variableregion nucleotide sequence (e.g., one or more genetically modified humanV_(H), D, and/or J_(H) gene segments), wherein the unrearranged heavychain variable region nucleotide sequence comprises an addition of atleast one histidine codon or a substitution of at least one endogenousnon-histidine codon with a histidine codon. In various embodiments, thegenome of the non-human animals comprises a modification (i) thatdeletes or renders nonfunctional all, or substantially all, endogenousimmunoglobulin V_(H), D, and/or J_(H) gene segments (e.g., via insertionof a nucleotide sequence, e.g., an exogenous nucleotide sequence, in theimmunoglobulin locus or via non-functional rearrangement or inversion ofendogenous V_(H), D, and/or J_(H) gene segments); and (ii) thatintroduces an unrearranged human heavy chain variable region nucleotidesequence (e.g., genetically modified human V_(H), D, or J_(H) genesegments), wherein the unrearranged heavy chain variable regionnucleotide sequence comprises an addition of at least one histidinecodon or a substitution of at least one endogenous non-histidine codonwith a histidine codon. In various embodiments, the unrearranged heavychain variable region nucleotide sequence is present at an endogenouslocus (i.e., where the unrearranged heavy chain variable regionnucleotide sequence is located in a wild-type non-human animal) orpresent ectopically (e.g., at a locus different from the endogenousimmunoglobulin heavy chain locus in its genome), or within itsendogenous locus (e.g., within an immunoglobulin variable locus, whereinthe endogenous locus is placed or moved to a different location in thegenome). In various embodiments, the immunoglobulin heavy chain variableregion nucleotide sequence is operably linked to a human or non-humanheavy chain constant region nucleotide sequence (e.g., a heavy chainconstant region nucleotide sequence that encodes an immunoglobulinisotype selected from IgM, IgD, IgA, IgE, and IgG).

Genetically modified non-human animals are provided that are capable ofexpressing a genetically modified immunoglobulin heavy variable domaincomprising one or more histidines, wherein the one or more histidinesare not encoded by a germline gene segment of a corresponding wild-typenon-human animal.

Genetically modified non-human animals are provided that comprise a Bcell population that is characterized by rearranged immunoglobulin heavychain variable genes that encode an immunoglobulin heavy chain variabledomain with one or more histidines that are not encoded by a germlinegene segment of a corresponding wild-type non-human animal.

Methods and compositions are provided for making non-human animals thatcomprise a genetically modified immunoglobulin heavy chain variablelocus comprising an unrearranged human heavy chain variable regionnucleotide sequence containing one or more histidine codons in at leastone reading frame that encodes a heavy chain variable domain.

Methods and compositions are provided for non-human animals that makeantigen-binding proteins that exhibit a pH-dependent binding of anantigen. Methods and compositions are provided for making non-humananimals that have B cell populations, or antibody populations, that areenriched (as compared with corresponding wild-type animals) withantigen-binding proteins that are pH-dependent, e.g., in particular,heavy chain variable domains, and/or antigen-binding fragments thereof.

In one aspect, a genetically modified immunoglobulin locus in a germlinegenome of a non-human animal is provided comprising an unrearrangedhuman heavy chain variable region nucleotide sequence, wherein theunrearranged heavy chain variable region nucleotide sequence comprisesan addition of least one histidine codon or a substitution of at leastone endogenous non-histidine codon with a histidine codon.

In one embodiment, the non-human animal is a mammal, including a rodent,e.g., a mouse, a rat, or a hamster.

In one embodiment, the added or substituted histidine codon is presentin an immunoglobulin heavy chain gene segment selected from a humanV_(H) gene segment, a human D gene segment, a human J_(H) gene segment,and a combination thereof. In one embodiment, the immunoglobulin heavychain gene segment is selected from a human germline V_(H) gene segment,a human germline D gene segment, a human germline J_(H) gene segment,and a combination thereof.

In one embodiment, the human V gene segment (V_(H)) is selected from thegroup consisting of V_(H)1-2, V_(H)1-3, V_(H)1-8, V_(H)1-18, V_(H)1-24,V_(H)1-45, V_(H)1-46, V_(H)1-58, V_(H)1-69, V_(H)2-5, V_(H)2-26,V_(H)2-70, V_(H)3-7, V_(H)3-9, V_(H)3-11, V_(H)3-13, V_(H)3-15,V_(H)3-16, V_(H)3-20, V_(H)3-21, V_(H)3-23, V_(H)3-30, V_(H)3-30-3,V_(H) 3-30-5, V_(H)3-33, V_(H)3-35, V_(H)3-38, V_(H)3-43, V_(H)3-48,V_(H)3-49, V_(H)3-53, V_(H)3-64, V_(H)3-66, V_(H)3-72, V_(H)3-73,V_(H)3-74, V_(H)4-4, V_(H)4-28, V_(H)4-30-1, V_(H)4-30-2, V_(H)4-30-4,V_(H)4-31, V_(H)4-34, V_(H)4-39, V_(H)4-59, V_(H)4-61, V_(H)5-51,V_(H)6-1, V_(H)7-4-1, V_(H)7-81, and a combination thereof.

In one embodiment, the human D gene segment is selected from the groupconsisting of D1-1, D1-7, D1-14, D1-20, D1-26, D2-2, D2-8, D2-15, D2-21,D3-3, D3-9, D3-10, D3-16, D3-22, D4-4, D4-11, D4-17, D4-23, D5-12, D5-5,D5-18, D5-24, D6-6, D6-13, D6-19, D6-25, D7-27, and a combinationthereof.

In one embodiment, the human J gene segment is selected from the groupconsisting of J_(H)1, J_(H)2, J_(H)3, J_(H)4, J_(H)5, J_(H)6, and acombination thereof.

In one embodiment, the added or substituted histidine codon is presentin the unrearranged heavy chain variable region nucleotide sequence thatencodes an N-terminal region, a loop 4 region, a CDR1, a CDR2, a CDR3,or a combination thereof.

In one embodiment, the unrearranged heavy chain variable regionnucleotide sequence comprises 2 or more, 3 or more, 4 or more, 5 ormore, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 ormore, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 ormore, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 ormore, 24 or more, or 25 or more, 26 or more, 27 or more, 28 or more, 29or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or more 35or more, 36 or more, 37 or more, 38 or more, 39 or more, 40 or more, 41or more, 42 or more, 43 or more, 44 or more, 45 or more, 46 or more, 47or more, 48 or more, 49 or more, 50 or more, 51 or more, 52 or more, 53or more, 54 or more, 55 or more, 56 or more, 57 or more, 58 or more, 59or more, 60 or more, or 61 or more of histidine codons.

In one embodiment, the unrearranged heavy chain variable regionnucleotide sequence is operably linked to a human or non-human heavychain constant region nucleotide sequence that encodes an immunoglobulinisotype selected from IgM, IgD, IgG, IgE, and IgA.

In one embodiment, the human unrearranged immunoglobulin heavy chainvariable region nucleotide sequence is operably linked to a human ornon-human heavy chain constant region nucleotide sequence selected froma C_(H)1, a hinge, a C_(H)2, a C_(H)3, and a combination thereof. In oneembodiment, the heavy chain constant region nucleotide sequencecomprises a C_(H)1, a hinge, a C_(H)2, and a C_(H)3 (i.e.,C_(H)1-hinge-C_(H)2-C_(H)3).

In one embodiment, a heavy chain constant region nucleotide sequence ispresent at an endogenous locus (i.e., where the nucleotide sequence islocated in a wild-type non-human animal) or present ectopically (e.g.,at a locus different from the endogenous immunoglobulin chain locus inits genome, or within its endogenous locus, e.g., within animmunoglobulin variable locus, wherein the endogenous locus is placed ormoved to a different location in the genome).

In one embodiment, the heavy chain constant region nucleotide sequencecomprises a modification in a C_(H)2 or a C_(H)3, wherein themodification increases the affinity of the heavy chain constant regionamino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a modification at position 250 (e.g., E or Q); 250 and 428(e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256(e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433(e.g., L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification atposition 250 and/or 428; or a modification at position 307 or 308 (e.g.,308F, V308F), and 434. In one embodiment, the modification comprises a428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I(e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K)and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y,254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Qand M428L); and a 307 and/or 308 modification (e.g., 308F or 308P),wherein the modification increases the affinity of the heavy chainconstant region amino acid sequence to FcRn in an acidic environment(e.g., in an endosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 252 and 257,wherein the modification increases the affinity of the human C_(H)2amino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 307 and 311,wherein the modification increases the affinity of the C_(H)2 amino acidsequence to FcRn in an acidic environment (e.g., in an endosome where pHranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)3 amino acid sequence, wherein the C_(H)3 aminoacid sequence comprises at least one modification between amino acidresidues at positions 433 and 436, wherein the modification increasesthe affinity of the C_(H)3 amino acid sequence to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,N434S, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,V259I, V308F, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising an N434A mutation.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M252Y,S254T, T256E, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of T250Q,M248L, or both.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of H433K,N434Y, or both.

In one embodiment, the genetically modified immunoglobulin locuscomprises: (1) a first allele, wherein the unrearranged humanimmunoglobulin heavy chain variable region nucleotide sequence asdescribed herein is operably linked to a first heavy chain constantregion nucleotide sequence encoding a first CH₃ amino acid sequence of ahuman IgG selected from IgG1, IgG2, IgG4, and a combination thereof; and(2) a second allele, wherein the unrearranged human immunoglobulin heavychain variable region nucleotide sequence as described herein isoperably linked to a second heavy chain constant region nucleotidesequence encoding a second C_(H)3 amino acid sequence of the human IgGselected from IgG1, IgG2, IgG4, and a combination thereof, and whereinthe second CH₃ amino acid sequence comprises a modification that reducesor eliminates binding for the second CH₃ amino acid sequence to ProteinA (see, for example, US 2010/0331527A1, which is incorporated byreference herein in its entirety).

In one embodiment, the second CH₃ amino acid sequence comprises an H95Rmodification (by IMGT exon numbering; H435R by EU numbering). In oneembodiment the second CH₃ amino acid sequence further comprises an Y96Fmodification (by IMGT exon numbering; H436F by EU). In anotherembodiment, the second CH₃ amino acid sequence comprises both an H95Rmodification (by IMGT exon numbering; H435R by EU numbering) and an Y96Fmodification (by IMGT exon numbering; H436F by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG1 and further comprises a mutation selected from the groupconsisting of D16E, L18M, N44S, K52N, V57M, and V82I (IMGT; D356E, L38M,N384S, K392N, V397M, and V422I by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG2 and further comprises a mutation selected from the groupconsisting of N44S, K52N, and V82I (IMGT: N384S, K392N, and V422I byEU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG4 and further comprises a mutation selected from the groupconsisting of Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (IMGT: Q355R,N384S, K392N, V397M, R409K, E419Q, and V422I by EU).

In one embodiment, the heavy chain constant region amino acid sequenceis a non-human constant region amino acid sequence, and the heavy chainconstant region amino acid sequence comprises one or more of any of thetypes of modifications described above.

In one embodiment, all or substantially all endogenous V_(H), D, andJ_(H) gene segments are deleted from an immunoglobulin heavy chain locusor rendered non-functional (e.g., via insertion of a nucleotide sequence(e.g., an exogenous nucleotide sequence) in the immunoglobulin locus orvia non-functional rearrangement, or inversion, of the endogenous V_(H),D, J_(H) segments). In one embodiment, e.g., about 80% or more, about85% or more, about 90% or more, about 95% or more, about 96% or more,about 97% or more, about 98% or more, or about 99% or more of allendogenous V_(H), D, or J_(H) gene segments are deleted or renderednon-functional. In one embodiment, e.g., at least 95%, 96%, 97%, 98%, or99% of endogenous functional V, D, or J gene segments are deleted orrendered non-functional.

In one embodiment, the genetically modified immunoglobulin heavy chainlocus comprises a modification that deletes or renders non-functionalall, or substantially all, endogenous V_(H), D, and J_(H) gene segments;and the genetically modified locus comprises an unrearranged heavy chainvariable region nucleotide sequence comprising one or more human V_(H),D, and/or J_(H) gene segments having one or more histidine codons,wherein the unrearranged heavy chain variable region nucleotide sequenceis present at an endogenous location (i.e., where the nucleotidesequence is located in a wild-type non-human animal) or presentectopically (e.g., at a locus different from the endogenousimmunoglobulin chain locus in its genome), or within its endogenouslocus (e.g., within an immunoglobulin variable locus, wherein theendogenous locus is placed or moved to a different location in thegenome).

In one embodiment, the genetically modified immunoglobulin locuscomprises an endogenous Adam6a gene, Adam6b gene, or both, and thegenetic modification does not affect the expression and/or function ofthe endogenous Adam6a gene, Adam6b gene, or both.

In one embodiment, the genetically modified immunoglobulin locuscomprises an ectopically present Adam6a gene, Adam6b gene, or both. Inone embodiment, the Adam6a gene is a non-human Adam6a gene. In oneembodiment, the Adam6a gene is a human Adam6a gene. In one embodiment,the Adam6b gene is a non-human Adam6b gene. In one embodiment, theAdam6b gene is a human Adam6b gene.

In one embodiment, the genetically modified immunoglobulin locus furthercomprises a humanized, unrearranged λ and/or κ light chain variable genesequence. In one embodiment, the humanized, unrearranged λ and/or κlight chain variable gene sequence is operably linked to animmunoglobulin light chain constant region nucleotide sequence selectedfrom a λ light chain constant region nucleotide sequence and a κ lightchain constant region nucleotide sequence. In one embodiment, thehumanized, unrearranged λ light chain variable region nucleotidesequence is operably linked to a λ light chain constant regionnucleotide sequence. In one embodiment, the λ light chain constantregion nucleotide sequence is a mouse, rat, or human sequence. In oneembodiment, the humanized, unrearranged κ light chain variable regionnucleotide sequence is operably linked to a κ light chain constantregion nucleotide sequence. In one embodiment, the κ light chainconstant region nucleotide sequence is a mouse, rat, or human sequence.

In one embodiment, the genetically modified immunoglobulin locuscomprises an unrearranged light chain variable gene sequence thatcontains at least one modification that introduces at least onehistidine codon in at least one reading frame encoding a light chainvariable domain. In one embodiment, the genetically modifiedimmunoglobulin locus comprises a rearranged (e.g., rearranged λ or κ V/Jsequence) sequence that comprises one, two, three, or four codons forhistidine in a light chain CDR. In one embodiment, the CDR is a selectedfrom a CDR1, CDR2, CDR3, and a combination thereof. In one embodiment,the unrearranged or rearranged light chain variable region nucleotidesequence is an unrearranged or rearranged human λ or κ light chainvariable region nucleotide sequence. In one embodiment, the unrearrangedor rearranged human λ or κ light chain variable region nucleotidesequence is present at an endogenous mouse immunoglobulin light chainlocus. In one embodiment, the mouse immunoglobulin light chain locus isa mouse κ locus. In one embodiment, the mouse immunoglobulin light chainlocus is a mouse λ locus.

In one embodiment, the genetically modified immunoglobulin locus asdescribed herein is present in an immunoglobulin heavy chain locus of amouse. In one embodiment, the genetically modified immunoglobulin locusis present in a humanized immunoglobulin heavy chain locus in aVELOCIMMUNE® mouse.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein exhibits a weaker antigen bindingat an acidic environment (e.g., at a pH of about 5.5 to about 6.0) thana corresponding wild-type heavy chain variable domain without thegenetic modification.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 2 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 25° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin heavy chain locus as described herein has adissociative half-life (t_(1/2)) of less than 2 min at an acidic pH(e.g., pH of about 5.5 to about 6.0) at 37° C. In one embodiment, anantigen-binding protein comprising a heavy chain variable domainexpressed by the genetically modified immunoglobulin heavy chain locusas described herein has a dissociative half-life (t_(1/2)) of less than1 min at an acidic pH (e.g., pH of about 5.5 to about 6.0) at 25° C. Inone embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 1 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 37° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin locus as described herein has at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold, at least about 25-fold, or at least about 30-fold decrease indissociative half-life (t_(1/2)) at an acidic pH (e.g., pH of about 5.5to about 6.0) as compared to the dissociative half-life (t_(1/2)) of theantigen-binding protein at a neutral pH (e.g., pH of about 7.0 to about7.4).

In one embodiment, the genetically modified immunoglobulin locusdescribed herein comprises a B cell population that, upon stimulationwith an antigen of interest, is capable of producing antigen-bindingproteins, e.g., antibodies, comprising a heavy chain variable domainwith one or more histidine residues. The antigen-binding proteins asdescribed herein, when administered into a subject, exhibits anincreased serum half-life over a corresponding wild-type antigen-bindingprotein, which possesses a similar or sufficiently similar amino acidsequence that encodes the heavy chain variable domain but does notcomprise a histidine residue in the heavy chain variable domain. In someembodiments, the antigen-binding protein described herein exhibits anincreased serum half-life that is at least about 2-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold higher than the corresponding wild-type antigen-binding protein,which possesses a similar or sufficiently similar amino acid sequencethat encodes the heavy chain variable domain but does not comprise ahistidine residue in the heavy chain variable domain.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinlocus as described herein is characterized by improved pH-dependentrecyclability, enhanced serum half-life, or both as compared with awild-type antigen-binding protein without the genetic modification asdescribed herein.

In one aspect, a genetically modified immunoglobulin locus in a germlinegenome of a non-human animal is provided comprising an unrearrangedhuman heavy chain variable region nucleotide sequence, wherein the humanunrearranged heavy chain variable region nucleotide sequence comprises asubstitution of at least one endogenous non-histidine codon with ahistidine codon.

In one embodiment, the non-human animal is a mammal, including a rodent,e.g., a mouse, a rat, or a hamster.

In one embodiment, 2 or more, 3 or more, 4 or more, 5 or more, 6 ormore, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 ormore, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 ormore, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 ormore, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 ormore, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 ormore, 37 or more, 38 or more, 39 or more, 40 or more, 41 or more, 42 ormore, 43 or more, 44 or more, 45 or more, 46 or more, 47 or more, 48 ormore, 49 or more, 50 or more, 51 or more, 52 or more, 53 or more, 54 ormore, 55 or more, 56 or more, 57 or more, 58 or more, 59 or more, 60 ormore, or 61 or more of the endogenous non-histidine codons are replacedwith histidine codons.

In one embodiment, the endogenous non-histone codon encodes the aminoacid selected from Y, N, D, Q, S, W, and R.

In one embodiment, the substituted histidine codon is present in anunrearranged heavy chain variable region nucleotide sequence thatencodes an immunoglobulin variable domain selected from an N-terminalregion, a loop 4 region, a CDR1, a CDR2, a CDR3, a combination thereof.

In one embodiment, the substituted histidine codon is present in anunrearranged heavy chain variable region nucleotide sequence thatencodes a complementary determining region (CDR) selected from a CDR1, aCDR2, a CDR3, and a combination thereof.

In one embodiment, the substituted histidine codon is present in anunrearranged heavy chain variable region nucleotide sequence thatencodes a frame region (FR) selected from FR1, FR2, FR3, FR4, and acombination thereof.

In one embodiment, the unrearranged heavy chain variable regionnucleotide sequence comprises a genetically modified human V_(H) genesegment, wherein one or more endogenous non-histidine codon in at leastone reading frame of the human V_(H) gene segment has been replaced witha histidine codon.

In one embodiment, the human unrearranged heavy chain variable regionnucleotide sequence comprises a modification that replaces at least oneendogenous non-histidine codon of a human V_(H) gene segment with ahistidine codon, wherein the human V_(H) gene segment is selected fromthe group consisting of V_(H)1-2, V_(H)1-3, V_(H)1-8, V_(H)1-18,V_(H)1-24, V_(H)1-45, V_(H)1-46, V_(H)1-58, V_(H)1-69, V_(H)2-5,V_(H)2-26, V_(H)2-70, V_(H)3-7, V_(H)3-9, V_(H)3-11, V_(H)3-13,V_(H)3-15, V_(H)3-16, V_(H)3-20, V_(H)3-21, V_(H)3-23, V_(H)3-30,V_(H)3-30-3, V_(H) 3-30-5, V_(H)3-33, V_(H)3-35, V_(H)3-38, V_(H)3-43,V_(H)3-48, V_(H)3-49, V_(H)3-53, V_(H)3-64, V_(H)3-66, V_(H)3-72,V_(H)3-73, V_(H)3-74, V_(H)4-4, V_(H)4-28, V_(H)4-30-1, V_(H)4-30-2,V_(H)4-30-4, V_(H)4-31, V_(H)4-34, V_(H)4-39, V_(H)4-59, V_(H)4-61,V_(H)5-51, V_(H)6-1, V_(H)7-4-1, V_(H)7-81, and a combination thereof.

In one embodiment, the human unrearranged heavy chain variable regionnucleotide sequence comprises a genetically modified human J_(H) genesegment, wherein one or more endogenous non-histidine codon in at leastone reading frame of the human J_(H) gene segment has been replaced witha histidine codon.

In one embodiment, the human unrearranged heavy chain variable regionnucleotide sequence comprises a modification that replaces at least oneendogenous non-histidine codon of a human J_(H) segment with a histidinecodon, wherein the human J_(H) gene segment is selected from the groupconsisting of J_(H)1, J_(H)2, 43, 44, J_(H)S, J_(H)6, and a combinationthereof.

In one embodiment, the substituted histidine codon is present in a heavychain variable region nucleotide sequence that encodes part of a CDR3.In one embodiment, the part of CDR3 comprises an amino acid sequencederived from a reading frame of a genetically modified human D genesegment comprising a modification that replaces at least one endogenousnon-histidine codon in the reading frame with a histidine codon.

In one embodiment, the endogenous non-histidine codon that issubstituted with a histidine codon encodes the amino acid selected fromY, N, D, Q, S, W, and R.

In one embodiment, the substituted histidine codon is present in atleast one reading frame of the human D gene segment that is mostfrequently observed in VELOCIMMUNE® humanized immunoglobulin mice.

In one embodiment, the reading frame of the genetically modified human Dgene segment that encodes part of CDR3 is selected from a hydrophobicframe, a stop frame, and a hydrophilic frame.

In one embodiment, the reading frame is a hydrophobic frame of a human Dgene segment.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D1-1 (GTTGT; SEQ ID NO: 88), D1-7(GITGT; SEQ ID NO: 89), D1-20 (GITGT; SEQ ID NO: 89), and D1-26 (GIVGAT;SEQ ID NO: 90), and the human D gene segment further comprises amodification that replaces at least one endogenous non-histidine codonin the nucleotide sequence with a histidine codon.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D2-2 (DIVVVPAAI; SEQ ID NO: 92),D2-8 (DIVLMVYAI; SEQ ID NO: 94), D2-15 (DIVVVVAAT; SEQ ID NO: 95), andD2-21 (HIVVVTAI; SEQ ID NO: 97), and the human D gene segment furthercomprises a modification that replaces at least one endogenousnon-histidine codon in the nucleotide sequence with a histidine codon.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D3-3 (ITIFGVVII; SEQ ID NO: 98),D3-9 (ITIF*LVII; SEQ ID NO: 99, SEQ ID NO:100), D3-10 (ITMVRGVII; SEQ IDNO:101), D3-16 (IMITFGGVIVI; SEQ ID NO:102), and D3-22 (ITMIVVVIT; SEQID NO:103), and the human D gene segment further comprises amodification that replaces at least one endogenous codon in thenucleotide sequence with a histidine codon.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D4-4 (TTVT; SEQ ID NO: 105), D4-11(TTVT; SEQ ID NO:105), D4-17 (TTVT; SEQ ID NO:105), D4-23 (TTVVT; SEQ IDNO: 106) and the human D gene segment further comprises a modificationthat replaces at least one endogenous non-histidine codon in thenucleotide sequence with a histidine codon.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D5-5 (VDTAMV; SEQ ID NO: 107),D5-12 (VDIVATI; SEQ ID NO: 108), D5-18 (VDTAMV; SEQ ID NO:107), andD5-24 (VEMATI; SEQ ID NO:109), and the human D gene segment furthercomprises a modification that replaces at least one endogenousnon-histidine codon in the nucleotide sequence with a histidine codon.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D6-6 (SIAAR; SEQ ID NO: 111),D6-13 (GIAAAG; SEQ ID NO: 113), and D6-19 (GIAVAG; SEQ ID NO:115), andthe human D gene segment further comprises a modification that replacesat least one endogenous non-histidine codon in the nucleotide sequencewith a histidine codon.

In one embodiment, the hydrophobic frame comprises a nucleotide sequencethat encodes human D7-27 (LTG), and the human D gene segment furthercomprises a modification that replaces at least one endogenousnon-histidine codon in the nucleotide sequence with a histidine codon.

In one embodiment, the reading frame is a stop reading frame of a humanD gene segment.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D1-1 (VQLER; SEQ ID NO:8),D1-7(V*LEL), D1-20(V*LER), D1-26 (V*WELL; SEQ ID NO: 12), and the humanD gene segment further comprises a modification that replaces at leastone endogenous non-histidine codon in the nucleotide sequence with ahistidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D2-2 (RIL**YQLLY; SEQ ID NO:14),D2-8 (RILY*WCMLY; SEQ ID NO:16 and SEQ ID NO: 17), D2-15 (RIL*WW*LLL),and D2-21 (SILWW*LLF; SEQ ID NO:19), and the human D gene segmentfurther comprises a modification that replaces at least one endogenousnon-histidine codon in the nucleotide sequence with a histidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D3-3 (VLRFLEWLLY; SEQ ID NO:21),D3-9 (VLRYFDWLL*; SEQ ID NO:23), D3-10 (VLLWFGELL*; SEQ ID NO:25), D3-16(VL*LRLGELSLY; SEQ ID NO:27), and D3-22 (VLL***WLLL; SEQ ID NO:29), andthe human D gene segment comprises a modification that replaces at leastone endogenous non-histidine codon in the nucleotide sequence with ahistidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D4-4 (*LQ*L), D4-11 (*LQ*L), D4-17(*LR*L), and D4-23 (*LRW*L), and the human D gene segment comprises amodification that replaces at least one endogenous non-histidine codonin the nucleotide sequence with a histidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D5-5 (WIQLWL; SEQ ID NO:35); D5-12(Wl*WLRL; SEQ ID NO:37), D5-18 (WIQLWL; SEQ ID NO:35), and D5-24(*RWLQL; SEQ ID NO:39), and the human D gene segment comprises amodification that replaces at least one endogenous non-histidine codonin the nucleotide sequence with a histidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D6-6 (V*QLV), D6-13 (V*QQLV; SEQID NO:41), and D6-19 (V*QWLV; SEQ ID NO:43), and the human D genesegment further comprises a modification that replaces at least oneendogenous non-histidine codon in the nucleotide sequence with ahistidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes D7-27 (*LG), and the humanD gene segment further comprises a modification that replaces at leastone endogenous codon of the human D gene segment in the nucleotidesequence with a histidine codon.

In one embodiment, the reading frame is a hydrophilic frame of a human Dgene segment.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D1-1 (YNWND; SEQ ID NO: 45), D1-7(YNWNY; SEQ ID NO: 47), D1-20 (YNWND; SEQ ID NO: 45), and D1-26 (YSGSYY;SEQ ID NO:49), and the human D gene segment further comprises amodification that replaces at least one endogenous codon in thenucleotide sequence with a histidine codon. In one embodiment, thehydrophilic frame comprises a nucleotide sequence that encodes the aminoacid sequence selected from the group consisting of SEQ ID NO: 46, SEQID NO: 48, SEQ ID NO: 50, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D2-2 (GYCSSTSCYT; SEQ ID NO:51),D2-8 (GYCTNGVCYT; SEQ ID NO: 53), D2-15 (GYCSGGSCYS; SEQ ID NO:55), andD2-21 (AYCGGDCYS; SEQ ID NO:57), and the human D gene segment furthercomprises a modification that replaces at least one endogenous codon inthe nucleotide sequence with a histidine codon. In one embodiment, thehydrophilic frame comprises a nucleotide sequence that encodes the aminoacid sequence selected from the group consisting of SEQ ID NO: 52, SEQID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D3-3 (YYDFWSGYYT; SEQ ID NO:59),D3-9 (YYDILTGYYN; SEQ ID NO:61), D3-10 (YYYGSGSYYN; SEQ ID NO:63), D3-16(YYDYVWGSYRYT; SEQ ID NO:65), and D3-22 (YYYDSSGYYY; SEQ ID NO:67), andthe human D gene segment further comprises a modification that replacesat least one endogenous codon in the nucleotide sequence with ahistidine codon. In one embodiment, the hydrophilic frame comprises anucleotide sequence encodes the amino acid sequence selected from thegroup consisting of SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ IDNO: 66, SEQ ID NO: 68, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D4-4 (DYSNY; SEQ ID NO:69), D4-11(DYSNY; SEQ ID NO:69), D4-17 (DYGDY; SEQ ID NO:71), and D4-23 (DYGGNS;SEQ ID NO:73), and the human D gene segment comprises a modificationthat replaces at least one endogenous codon in the nucleotide sequencewith a histidine codon. In one embodiment, the hydrophilic framecomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of SEQ ID NO: 70, SEQ ID NO: 72, SEQID NO: 74, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D5-5 (GYSYGY; SEQ ID NO:75), D5-12(GYSGYDY; SEQ ID NO:77), D5-18 (GYSYGY; SEQ ID NO:75), and D5-24(RDGYNY; SEQ ID NO:79), and the human D gene segment further comprises amodification that replaces at least one endogenous codon in thenucleotide sequence with a histidine codon. In one embodiment, thehydrophilic frame comprises a nucleotide sequence that encodes the aminoacid sequence selected from the group consisting of SEQ ID NO: 76, SEQID NO: 78, SEQ ID NO: 80, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D6-6 (EYSSSS; SEQ ID NO: 81),D6-13 (GYSSSWY; SEQ ID NO:83), and D6-19 (GYSSGWY; SEQ ID NO:85), andthe human D gene segment further comprises a modification that replacesat least one endogenous codon in the nucleotide sequence with ahistidine codon. In one embodiment, the hydrophilic frame comprises anucleotide sequence that encodes the amino acid sequence selected fromthe group consisting of SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQID NO: 76, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes D7-27 (NWG), and the humanD gene segment further comprises a modification that replaces at leastone endogenous codon in the nucleotide sequence a histidine codon.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 48, SEQID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO:68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ IDNO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, anda combination thereof.

In one embodiment, the human unrearranged immunoglobulin heavy chainvariable region nucleotide sequence is operably linked to a human ornon-human heavy chain constant region nucleotide sequence selected froma C_(H)1, a hinge, a C_(H)2, a C_(H)3, and a combination thereof. In oneembodiment, the heavy chain constant region nucleotide sequencecomprises a C_(H)1, a hinge, a C_(H)2, and a C_(H)3 (i.e.,C_(H)1-hinge-C_(H)2-C_(H)3).

In one embodiment, a heavy chain constant region nucleotide sequence ispresent at an endogenous locus (i.e., where the nucleotide sequence islocated in a wild-type non-human animal) or present ectopically (e.g.,at a locus different from the endogenous immunoglobulin chain locus inits genome), or within its endogenous locus, e.g., within animmunoglobulin variable locus, wherein the endogenous locus is placed ormoved to a different location in the genome.

In one embodiment, the heavy chain constant region nucleotide sequencecomprises a modification in a C_(H)2 or a C_(H)3, wherein themodification increases the affinity of the heavy chain constant regionamino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a modification at position 250 (e.g., E or Q); 250 and 428(e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256(e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433(e.g., L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification atposition 250 and/or 428; or a modification at position 307 or 308 (e.g.,308F, V308F), and 434. In one embodiment, the modification comprises a428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I(e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K)and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y,254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Qand M428L); and a 307 and/or 308 modification (e.g., 308F or 308P),wherein the modification increases the affinity of the heavy chainconstant region amino acid sequence to FcRn in an acidic environment(e.g., in an endosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 252 and 257,wherein the modification increases the affinity of the human C_(H)2amino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 307 and 311,wherein the modification increases the affinity of the C_(H)2 amino acidsequence to FcRn in an acidic environment (e.g., in an endosome where pHranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)3 amino acid sequence, wherein the C_(H)3 aminoacid sequence comprises at least one modification between amino acidresidues at positions 433 and 436, wherein the modification increasesthe affinity of the C_(H)3 amino acid sequence to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,N434S, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,V259I, V308F, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising an N434A mutation.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M252Y,S254T, T256E, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of T250Q,M248L, or both.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of H433K,N434Y, or both.

In one embodiment, the genetically modified immunoglobulin locuscomprises: (1) a first allele, wherein the unrearranged humanimmunoglobulin heavy chain variable region nucleotide sequence asdescribed herein is operably linked to a first heavy chain constantregion nucleotide sequence encoding a first CH₃ amino acid sequence of ahuman IgG selected from IgG1, IgG2, IgG4, and a combination thereof; and(2) a second allele, wherein the unrearranged human immunoglobulin heavychain variable region nucleotide sequence as described herein isoperably linked to a second heavy chain constant region nucleotidesequence encoding a second C_(H)3 amino acid sequence of the human IgGselected from IgG1, IgG2, IgG4, and a combination thereof, and whereinthe second CH₃ amino acid sequence comprises a modification that reducesor eliminates binding for the second CH₃ amino acid sequence to ProteinA (see, for example, US 2010/0331527A1, which is incorporated byreference herein in its entirety).

In one embodiment, the second CH₃ amino acid sequence comprises an H95Rmodification (by IMGT exon numbering; H435R by EU numbering). In oneembodiment the second CH₃ amino acid sequence further comprises an Y96Fmodification (by IMGT exon numbering; H436F by EU). In anotherembodiment, the second CH₃ amino acid sequence comprises both an H95Rmodification (by IMGT exon numbering; H435R by EU numbering) and an Y96Fmodification (by IMGT exon numbering; H436F by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG1 and further comprises a mutation selected from the groupconsisting of D16E, L18M, N44S, K52N, V57M, and V82I (IMGT; D356E, L38M,N384S, K392N, V397M, and V422I by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG2 and further comprises a mutation selected from the groupconsisting of N44S, K52N, and V82I(IMGT: N384S, K392N, and V422I by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG4 and further comprises a mutation selected from the groupconsisting of Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (IMGT: Q355R,N384S, K392N, V397M, R409K, E419Q, and V422I by EU).

In one embodiment, the heavy chain constant region amino acid sequenceis a non-human constant region amino acid sequence, and the heavy chainconstant region amino acid sequence comprises one or more of any of thetypes of modifications described above.

In one embodiment, the heavy chain constant region nucleotide sequenceis a human heavy chain constant region amino acid sequence, and thehuman heavy chain constant region amino acid sequence comprises one ormore of any of the types of modifications described above.

In one embodiment, all or substantially all endogenous V_(H), D, andJ_(H) gene segments are deleted from an immunoglobulin heavy chain locusor rendered non-functional (e.g., via insertion of a nucleotidesequence, e.g., an exogenous nucleotide sequence, in the immunoglobulinlocus or via non-functional rearrangement, or inversion, of theendogenous V_(H), D, J_(H) segments). In one embodiment, e.g., about 80%or more, about 85% or more, about 90% or more, about 95% or more, about96% or more, about 97% or more, about 98% or more, or about 99% or moreof all endogenous V_(H), D, or J_(H) gene segments are deleted orrendered non-functional. In one embodiment, e.g., at least 95%, 96%,97%, 98%, or 99% of endogenous functional V, D, or J gene segments aredeleted or rendered non-functional.

In one embodiment, the genetically modified locus comprises amodification that deletes or renders non-functional all or substantiallyall endogenous V_(H), D, and J_(H) gene segments; and the genomic locuscomprises a genetically modified, unrearranged human heavy chainvariable region nucleotide sequence comprising a substitution of atleast one endogenous non-histidine codon with a histidine codon in atleast one reading frame. In one embodiment, the genetically modified,unrearranged immunoglobulin heavy chain variable gene sequence ispresent at an endogenous location (i.e., where the nucleotide sequenceis located in a wild-type non-human animal) or present ectopically(e.g., at a locus different from the endogenous immunoglobulin chainlocus in its genome), or within its endogenous locus, e.g., within animmunoglobulin variable locus, wherein the endogenous locus is placed ormoved to a different location in the genome.

In one embodiment, the genetically modified locus comprises anendogenous Adam6a gene, Adam6b gene, or both, and the geneticmodification does not affect the expression and/or function of theendogenous Adam6a gene, Adam6b gene, or both.

In one embodiment, the genetically modified locus comprises anectopically present Adam6a gene, Adam6b gene, or both. In oneembodiment, the Adam6a gene is a non-human Adam6a gene. In oneembodiment, the Adam6a gene is a mouse Adam6a gene. In one embodiment,the Adam6a gene is a human Adam6a gene. In one embodiment, the Adam6bgene is a non-human Adam6b gene. In one embodiment, the Adam6b gene is amouse Adam6b gene. In one embodiment, the Adam6b gene is a human Adam6bgene.

In one embodiment, the genetically modified immunoglobulin locus furthercomprises a humanized, unrearranged λ and/or κ light chain variable genesequence. In one embodiment, the humanized, unrearranged λ and/or κlight chain variable gene sequence is operably linked to animmunoglobulin light chain constant region nucleotide sequence selectedfrom a λ light chain constant region nucleotide sequence and a κ lightchain constant region nucleotide sequence. In one embodiment, thehumanized, unrearranged λ light chain variable region nucleotidesequence is operably linked to a λ light chain constant regionnucleotide sequence. In one embodiment, the λ light chain constantregion nucleotide sequence is a mouse, rat, or human sequence. In oneembodiment, the humanized, unrearranged κ light chain variable regionnucleotide sequence is operably linked to a κ light chain constantregion nucleotide sequence. In one embodiment, the κ light chainconstant region nucleotide sequence is a mouse, rat, or human sequence.

In one embodiment, the genetically modified immunoglobulin locuscomprises an unrearranged light chain variable gene sequence thatcontains at least one modification that introduces at least onehistidine codon in at least one reading frame encoding a light chainvariable domain. In one embodiment, the genetically modifiedimmunoglobulin locus comprises a rearranged (e.g., a rearranged λ or κV/J sequence) sequence that comprises one, two, three, or four codonsfor histidine in a light chain CDR. In one embodiment, the CDR is aselected from a CDR1, CDR2, CDR3, and a combination thereof. In oneembodiment, the unrearranged or rearranged light chain variable regionnucleotide sequence is an unrearranged or rearranged human λ or κ lightchain variable region nucleotide sequence. In one embodiment, theunrearranged or rearranged human λ or κ light chain variable regionnucleotide sequence is present at an endogenous mouse immunoglobulinlight chain locus. In one embodiment, the mouse immunoglobulin lightchain locus is a mouse κ locus. In one embodiment the mouseimmunoglobulin light chain locus is a mouse λ locus.

In one embodiment, the genetically modified immunoglobulin locus asdescribed herein is present in an immunoglobulin heavy chain locus of amouse. In one embodiment, the genetically modified immunoglobulin locusis present in a humanized immunoglobulin heavy chain locus in aVELOCIMMUNE® mouse.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein exhibits a weaker antigen bindingat an acidic environment (e.g., at a pH of about 5.5 to about 6.0) thana corresponding wild-type heavy chain variable domain without thegenetic modification described herein.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 2 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 25° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin heavy chain locus as described herein has adissociative half-life (t_(1/2)) of less than 2 min at an acidic pH(e.g., pH of about 5.5 to about 6.0) at 37° C. In one embodiment, anantigen-binding protein comprising a heavy chain variable domainexpressed by the genetically modified immunoglobulin heavy chain locusas described herein has a dissociative half-life (t_(1/2)) of less than1 min at an acidic pH (e.g., pH of about 5.5 to about 6.0) at 25° C. Inone embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 1 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 37° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin locus as described herein has at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold, at least about 25-fold, or at least about 30-fold decrease indissociative half-life (t_(1/2)) at an acidic pH (e.g., pH of about 5.5to about 6.0) as compared to the dissociative half-life (t_(1/2)) of theantigen-binding protein at a neutral pH (e.g., pH of about 7.0 to about7.4).

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinlocus as described herein is characterized by improved pH-dependentrecyclability, enhanced serum half-life, or both as compared with awild-type antigen-binding protein without the genetic modification.

In one embodiment, the genetically modified immunoglobulin locusdescribed herein comprises a B cell population that, upon stimulationwith an antigen of interest, is capable of producing antigen-bindingproteins, e.g., antibodies, comprising a heavy chain variable domaincomprising one or more histidine residues. The antigen-binding proteins,which are produced by the genetically modified immunoglobulin locusdescribed herein, when administered into a subject, exhibits anincreased serum half-life over a corresponding wild-type antigen-bindingprotein, which possesses a similar or sufficiently similar amino acidsequence that encodes the heavy chain variable domain but does notcomprise a histidine residue in the heavy chain variable domain. In someembodiments, the antigen-binding protein described herein exhibits anincreased serum half-life that is at least about 2-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold higher than the corresponding wild-type antigen-binding protein,which possesses a similar or sufficiently similar amino acid sequencethat encodes the heavy chain variable domain but does not comprise ahistidine residue in the heavy chain variable domain.

In one aspect, a genetically modified immunoglobulin locus of anon-human animal comprising a human V_(H), D, and J_(H) gene segment isprovided, wherein at least one human D gene segment has been inverted 5′to 3′ with respect to a corresponding wild-type sequence, and wherein atleast one reading frame of the inverted human D gene segment comprisesone ore more histidine codon.

In one embodiment, the non-human animal is a mammal, including a rodent,e.g., a mouse, a rat, or a hamster

In one embodiment, the genetically modified immunoglobulin locus ispresent in a germline genome.

In one embodiment, the genetically modified immunoglobulin locus encodesan immunoglobulin heavy chain variable domain comprising one or more, 2or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 ormore, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 ormore, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 ormore, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 ormore, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 ormore, 33 or more, or 34 or more of histidine residues.

In one embodiment, at least two, at least three, at least four, at leastfive, at least six, at least seven, at least eight, at least nine, atleast ten, at least eleven, at least twelve, at least thirteen, at leastfourteen, at least fifteen, at least sixteen, at least seventeen, atleast eighteen, at least nineteen, at least twenty, at least twenty one,at least twenty two, at least twenty three, at least twenty four, or allor substantially all of functional human D gene segments have invertedorientation with respect to corresponding wild type sequences.

In one embodiment, all or substantially all of endogenous immunoglobulinV_(H), D, J_(H) gene segments are deleted from the immunoglobulin heavychain locus or rendered non-functional (e.g., via insertion of anucleotide sequence, e.g., exogenous nucleotide sequence, in theimmunoglobulin locus or via non-functional rearrangement or inversion ofall, or substantially all, endogenous immunoglobulin V_(H), D, J_(H)segments), and the genetically modified immunoglobulin locus comprises ahuman V_(H), D, and J_(H) gene segments, wherein at least one human Dgene segment is present in an inverted orientation with respect to acorresponding wild type sequence, and wherein at least one reading framein the inverted human D gene segment comprises at least one histidinecodon.

In one embodiment, the inverted human D gene segment is operably linkedto a human V_(H) gene segment, and/or human J_(H) gene segment

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence isselected from the group consisting of D1-1, D1-7, D1-20, D1-26, D2-2,D2-8, D2-15, D2-21, D3-3, D3-9, D3-10, D3-16, D3-22, D4-4, D4-11, D4-17,D4-23, D5-5, D5-12, D5-18, D5-24, D6-6, D6-13, D6-19, D7-27, and acombination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD1 gene segment selected from the group consisting of D1-1, D1-7, D1-20,D1-26, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative a corresponding wild type sequence is a D2gene segment selected from the group consisting of D2-2, D2-8, D2-15,D2-21, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD3 gene segment selected from the group consisting of D3-3, D3-9, D3-10,D3-16, D3-22, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD4 gene segment selected from the group consisting of D4-4, D4-11,D4-17, D4-23, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD5 gene segment selected from the group consisting of D5-5, D5-12,D5-18, D5-24, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD6 gene segment selected from the group consisting of D6-6, D6-13,D6-19, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence isD7-27.

In one embodiment, the reading frame of the human D gene segment isselected from a stop reading frame, a hydrophilic reading frame, and ahydrophobic reading frame, and at least one reading frame of theinverted human D gene segment comprises one or more histidine codon.

In one embodiment, the unrearranged heavy chain variable regionnucleotide sequence comprising the inverted human D gene segment isoperably linked to a human or non-human heavy chain constant regionnucleotide sequence that encodes an immunoglobulin isotype selected fromIgM, IgD, IgG, IgE, and IgA.

In one embodiment, the unrearranged heavy chain variable regionnucleotide sequence comprising the inverted human D gene segment isoperably linked to a human or non-human heavy chain constant regionnucleotide sequence that encodes an immunoglobulin isotype selected fromIgM, IgD, IgG, IgE, and IgA.

In one embodiment, the human unrearranged immunoglobulin heavy chainvariable region nucleotide sequence is operably linked to a human ornon-human heavy chain constant region nucleotide sequence selected froma C_(H)1, a hinge, a C_(H)2, a C_(H)3, and a combination thereof. In oneembodiment, the heavy chain constant region nucleotide sequencecomprises a C_(H)1, a hinge, a C_(H)2, and a C_(H)3 (i.e.,C_(H)1-hinge-C_(H)2-C_(H)3).

In one embodiment, a heavy chain constant region nucleotide sequence ispresent at an endogenous locus (i.e., where the nucleotide sequence islocated in a wild-type non-human animal) or present ectopically (e.g.,at a locus different from the endogenous immunoglobulin chain locus inits genome), or within its endogenous locus, e.g., within animmunoglobulin variable locus, wherein the endogenous locus is placed ormoved to a different location in the genome.

In one embodiment, the heavy chain constant region nucleotide sequencecomprises a modification in a C_(H)2 or a C_(H)3, wherein themodification increases the affinity of the heavy chain constant regionamino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a modification at position 250 (e.g., E or Q); 250 and 428(e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256(e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433(e.g., L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification atposition 250 and/or 428; or a modification at position 307 or 308 (e.g.,308F, V308F), and 434. In one embodiment, the modification comprises a428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I(e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K)and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y,254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Qand M428L); and a 307 and/or 308 modification (e.g., 308F or 308P),wherein the modification increases the affinity of the heavy chainconstant region amino acid sequence to FcRn in an acidic environment(e.g., in an endosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 252 and 257,wherein the modification increases the affinity of the human C_(H)2amino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 307 and 311,wherein the modification increases the affinity of the C_(H)2 amino acidsequence to FcRn in an acidic environment (e.g., in an endosome where pHranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)3 amino acid sequence, wherein the C_(H)3 aminoacid sequence comprises at least one modification between amino acidresidues at positions 433 and 436, wherein the modification increasesthe affinity of the C_(H)3 amino acid sequence to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,N434S, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,V259I, V308F, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising an N434A mutation.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M252Y,S254T, T256E, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of T250Q,M248L, or both.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of H433K,N434Y, or both.

In one embodiment, the genetically modified immunoglobulin locuscomprises: (1) a first allele, wherein the unrearranged humanimmunoglobulin heavy chain variable region nucleotide sequence asdescribed herein is operably linked to a first heavy chain constantregion nucleotide sequence encoding a first CH₃ amino acid sequence of ahuman IgG selected from IgG1, IgG2, IgG4, and a combination thereof; and(2) a second allele, wherein the unrearranged human immunoglobulin heavychain variable region nucleotide sequence as described herein isoperably linked to a second heavy chain constant region nucleotidesequence encoding a second C_(H)3 amino acid sequence of the human IgGselected from IgG1, IgG2, IgG4, and a combination thereof, and whereinthe second CH₃ amino acid sequence comprises a modification that reducesor eliminates binding for the second CH₃ amino acid sequence to ProteinA (see, for example, US 2010/0331527A1, incorporated by reference hereinin its entirety).

In one embodiment, the second CH₃ amino acid sequence comprises an H95Rmodification (by IMGT exon numbering; H435R by EU numbering). In oneembodiment the second CH₃ amino acid sequence further comprises an Y96Fmodification (by IMGT exon numbering; H436F by EU). In anotherembodiment, the second CH₃ amino acid sequence comprises both an H95Rmodification (by IMGT exon numbering; H435R by EU numbering) and an Y96Fmodification (by IMGT exon numbering; H436F by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG1 and further comprises a mutation selected from the groupconsisting of D16E, L18M, N44S, K52N, V57M, and V82I (IMGT; D356E, L38M,N384S, K392N, V397M, and V422I by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG2 and further comprises a mutation selected from the groupconsisting of N44S, K52N, and V82I (IMGT: N384S, K392N, and V422I byEU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG4 and further comprises a mutation selected from the groupconsisting of Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (IMGT: Q355R,N384S, K392N, V397M, R409K, E419Q, and V422I by EU).

In one embodiment, the heavy chain constant region amino acid sequenceis a non-human constant region amino acid sequence, and the heavy chainconstant region amino acid sequence comprises one or more of any of thetypes of modifications described above.

In one embodiment, the heavy chain constant region nucleotide sequenceis a human heavy chain constant region amino acid sequence, and thehuman heavy chain constant region amino acid sequence comprises one ormore of any of the types of modifications described above.

In one embodiment, all, or substantially all, endogenous V_(H), D, andJ_(H) gene segments are deleted from an immunoglobulin heavy chain locusor rendered non-functional (e.g., via insertion of a nucleotide sequence(e.g., an exogenous nucleotide sequence) in the immunoglobulin locus orvia non-functional rearrangement, or inversion, of the endogenous V_(H),D, J_(H) segments). In one embodiment, e.g., about 80% or more, about85% or more, about 90% or more, about 95% or more, about 96% or more,about 97% or more, about 98% or more, or about 99% or more of allendogenous V_(H), D, or J_(H) gene segments are deleted or renderednon-functional. In one embodiment, e.g., at least 95%, 96%, 97%, 98%, or99% of endogenous functional V, D, or J gene segments are deleted orrendered non-functional.

In one embodiment, the genetically modified immunoglobulin heavy chainlocus comprises a modification that deletes or renders non-functional,all or substantially all, endogenous V_(H), D, and J_(H) gene segments;and the genetically modified locus comprises an unrearranged heavy chainvariable region nucleotide sequence comprising at least one invertedhuman D gene segment as described herein wherein the unrearranged heavychain variable region nucleotide sequence is present at an endogenouslocation (i.e., where the nucleotide sequence is located in a wild-typenon-human animal) or present ectopically (e.g., at a locus differentfrom the endogenous immunoglobulin chain locus in its genome, or withinits endogenous locus, e.g., within an immunoglobulin variable locus,wherein the endogenous locus is placed or moved to a different locationin the genome).

In one embodiment, the genetically modified immunoglobulin locuscomprises an endogenous Adam6a gene, Adam6b gene, or both, and thegenetic modification does not affect the expression and/or function ofthe endogenous Adam6a gene, Adam6b gene, or both.

[000163] In one embodiment, the genetically modified immunoglobulinlocus comprises an ectopically present Adam6a gene, Adam6b gene, orboth. In one embodiment, the Adam6a gene is a non-human Adam6a gene. Inone embodiment, the Adam6a gene is a mouse Adam6a gene. In oneembodiment, the Adam6a gene is a human Adam6a gene. In one embodiment,the Adam6b gene is a non-human Adam6b gene. In one embodiment, theAdam6b gene is a mouse Adam6b gene. In one embodiment, the Adam6b geneis a human Adam6b gene.

In one embodiment, the genetically modified immunoglobulin locus furthercomprises a humanized, unrearranged λ and/or κ light chain variable genesequence. In one embodiment, the humanized, unrearranged λ and/or κlight chain variable gene sequence is operably linked to animmunoglobulin light chain constant region nucleotide sequence selectedfrom a λ light chain constant region nucleotide sequence and a κ lightchain constant region nucleotide sequence. In one embodiment, thehumanized, unrearranged λ light chain variable region nucleotidesequence is operably linked to a λ light chain constant regionnucleotide sequence. In one embodiment, the λ light chain constantregion nucleotide sequence is a mouse, rat, or human sequence. In oneembodiment, the humanized, unrearranged κ light chain variable regionnucleotide sequence is operably linked to a κ light chain constantregion nucleotide sequence. In one embodiment, the κ light chainconstant region nucleotide sequence is a mouse, rat, or human sequence.

In one embodiment, the genetically modified immunoglobulin locuscomprises an unrearranged light chain variable gene sequence thatcontains at least one modification that introduces at least onehistidine codon in at least one reading frame encoding a light chainvariable domain. In one embodiment, the genetically modifiedimmunoglobulin locus comprises a rearranged (e.g., a rearranged λ or κV/J sequence) sequence that comprises one, two, three, or four codonsfor histidine in a light chain CDR. In one embodiment, the CDR is aselected from a CDR1, CDR2, CDR3, and a combination thereof. In oneembodiment, the unrearranged or rearranged light chain variable regionnucleotide sequence is an unrearranged or rearranged human λ or κ lightchain variable region nucleotide sequence. In one embodiment, theunrearranged or rearranged human λ or κ light chain variable regionnucleotide sequence is present at an endogenous mouse immunoglobulinlight chain locus. In one embodiment, the mouse immunoglobulin lightchain locus is a mouse κ locus. In one embodiment, the mouseimmunoglobulin light chain locus is a mouse immunoglobulin light chainlocus is a mouse λ locus.

In one embodiment, the genetically modified immunoglobulin locus asdescribed herein is present in an immunoglobulin heavy chain locus of amouse. In one embodiment, the genetically modified immunoglobulin locusis present in a humanized immunoglobulin heavy chain locus in aVELOCIMMUNE® mouse.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein exhibits a weaker antigen bindingat an acidic environment (e.g., at a pH of about 5.5 to about 6.0) thana corresponding wild-type heavy chain variable domain without thegenetic modification.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 2 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 25° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin heavy chain locus as described herein has adissociative half-life (t_(1/2)) of less than 2 min at an acidic pH(e.g., pH of about 5.5 to about 6.0) at 37° C. In one embodiment, anantigen-binding protein comprising a heavy chain variable domainexpressed by the genetically modified immunoglobulin heavy chain locusas described herein has a dissociative half-life (t_(1/2)) of less than1 min at an acidic pH (e.g., pH of about 5.5 to about 6.0) at 25° C. Inone embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 1 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 37° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin locus as described herein has at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold, at least about 25-fold, or at least about 30-fold decrease indissociative half-life (t_(1/2)) at an acidic pH (e.g., pH of about 5.5to about 6.0) as compared to the dissociative half-life (t_(1/2)) of theantigen-binding protein at a neutral pH (e.g., pH of about 7.0 to about7.4).

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinlocus as described herein is characterized by improved pH-dependentrecyclability, enhanced serum half-life, or both as compared with awild-type antigen-binding protein without the genetic modification.

In one embodiment, the genetically modified immunoglobulin locusdescribed herein comprises a B cell population that, upon stimulationwith an antigen of interest, is capable of producing antigen-bindingproteins, e.g., antibodies, comprising a heavy chain variable domaincomprising one or more histidine residues. The antigen-binding proteinsas described herein when administered into a subject, exhibits anincreased serum half-life over a corresponding wild-type antigen-bindingprotein, which possesses a similar or sufficiently similar amino acidsequence that encodes the heavy chain variable domain but does notcomprise a histidine residue in the heavy chain variable domain. In someembodiments, the antigen-binding protein described herein exhibits anincreased serum half-life that is at least about 2-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold higher than the corresponding wild-type antigen-binding protein,which possesses a similar or sufficiently similar amino acid sequencethat encodes the heavy chain variable domain but does not comprise ahistidine residue in the heavy chain variable domain.

In one aspect, a non-human animal is provided comprising in its germlinegenome a genetically modified immunoglobulin locus comprising anunrearranged human heavy chain variable region nucleotide sequence,wherein the unrearranged heavy chain variable region nucleotide sequencecomprises an addition of least one histidine codon or a substitution ofat least one endogenous non-histidine codon with a histidine codon.

In one embodiment, the non-human animal is a mammal, including a rodent,e.g., a mouse, a rat, or a hamster.

In one embodiment, the added or substituted histidine codon is presentin an immunoglobulin heavy chain gene segment selected from a humanV_(H) gene segment, a human D gene segment, a human J_(H) gene segment,and a combination thereof. In one embodiment, the immunoglobulin heavychain gene segment is selected from a human germline V_(H) gene segment,a human germline D gene segment, a human germline J_(H) gene segment,and a combination thereof.

In one embodiment, the human V_(H) gene segment is selected from thegroup consisting of V_(H)1-2, V_(H)1-3, V_(H)1-8, V_(H)1-18, V_(H)1-24,V_(H)1-45, V_(H)1-46, V_(H)1-58, V_(H)1-69, V_(H)2-5, V_(H)2-26,V_(H)2-70, V_(H)3-7, V_(H)3-9, V_(H)3-11, V_(H)3-13, V_(H)3-15,V_(H)3-16, V_(H)3-20, V_(H)3-21, V_(H)3-23, V_(H)3-30, V_(H)3-30-3,V_(H) 3-30-5, V_(H)3-33, V_(H)3-35, V_(H)3-38, V_(H)3-43, V_(H)3-48,V_(H)3-49, V_(H)3-53, V_(H)3-64, V_(H)3-66, V_(H)3-72, V_(H)3-73,V_(H)3-74, V_(H)4-4, V_(H)4-28, V_(H)4-30-1, V_(H)4-30-2, V_(H)4-30-4,V_(H)4-31, V_(H)4-34, V_(H)4-39, V_(H)4-59, V_(H)4-61, V_(H)5-51,V_(H)6-1, V_(H)7-4-1, V_(H)7-81, and a combination thereof.

In one embodiment, the human D gene segment is selected from the groupconsisting of D1-1, D1-7, D1-14, D1-20, D1-26, D2-2, D2-8, D2-15, D2-21,D3-3, D3-9, D3-10, D3-16, D3-22, D4-4, D4-11, D4-17, D4-23, D5-12, D5-5,D5-18, D5-24, D6-6, D6-13, D6-19, D6-25, D7-27, and a combinationthereof.

In one embodiment, the human J_(H) gene segment is selected from thegroup consisting of J_(H)1, J_(H)2, J_(H)3, J_(H)4, J_(H)5, J_(H)6, anda combination thereof.

In one embodiment, the added or substituted histidine codon is presentin the unrearranged heavy chain variable region nucleotide sequenceencoding an N-terminal region, a loop 4 region, a CDR1, a CDR2, a CDR3,or a combination thereof.

In one embodiment, the unrearranged heavy chain variable regionnucleotide sequence comprises 2 or more, 3 or more, 4 or more, 5 ormore, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 ormore, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 ormore, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 ormore, 24 or more, or 25 or more, 26 or more, 27 or more, 28 or more, 29or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or more 35or more, 36 or more, 37 or more, 38 or more, 39 or more, 40 or more, 41or more, 42 or more, 43 or more, 44 or more, 45 or more, 46 or more, 47or more, 48 or more, 49 or more, 50 or more, 51 or more, 52 or more, 53or more, 54 or more, 55 or more, 56 or more, 57 or more, 58 or more, 59or more, 60 or more, or 61 or more of histidine codons.

In one embodiment, the unrearranged heavy chain variable regionnucleotide sequence comprising the inverted human D gene segment isoperably linked to a human or non-human heavy chain constant regionnucleotide sequence that encodes an immunoglobulin isotype selected fromIgM, IgD, IgG, IgE, and IgA.

In one embodiment, the human unrearranged immunoglobulin heavy chainvariable region nucleotide sequence is operably linked to a human ornon-human heavy chain constant region nucleotide sequence selected froma C_(H)1, a hinge, a C_(H)2, a C_(H)3, and a combination thereof. In oneembodiment, the heavy chain constant region nucleotide sequencecomprises a C_(H)1, a hinge, a C_(H)2, and a C_(H)3 (i.e.,C_(H)1-hinge-C_(H)2-C_(H)3).

In one embodiment, a heavy chain constant region nucleotide sequence ispresent at an endogenous locus (i.e., where the nucleotide sequence islocated in a wild-type non-human animal) or present ectopically (e.g.,at a locus different from the endogenous immunoglobulin chain locus inits genome), or within its endogenous locus, e.g., within animmunoglobulin variable locus, wherein the endogenous locus is placed ormoved to a different location in the genome.

In one embodiment, the heavy chain constant region nucleotide sequencecomprises a modification in a C_(H)2 or a C_(H)3, wherein themodification increases the affinity of the heavy chain constant regionamino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a modification at position 250 (e.g., E or Q); 250 and 428(e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256(e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433(e.g., L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification atposition 250 and/or 428; or a modification at position 307 or 308 (e.g.,308F, V308F), and 434. In one embodiment, the modification comprises a428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I(e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K)and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y,254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Qand M428L); and a 307 and/or 308 modification (e.g., 308F or 308P),wherein the modification increases the affinity of the heavy chainconstant region amino acid sequence to FcRn in an acidic environment(e.g., in an endosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 252 and 257,wherein the modification increases the affinity of the human C_(H)2amino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 307 and 311,wherein the modification increases the affinity of the C_(H)2 amino acidsequence to FcRn in an acidic environment (e.g., in an endosome where pHranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)3 amino acid sequence, wherein the C_(H)3 aminoacid sequence comprises at least one modification between amino acidresidues at positions 433 and 436, wherein the modification increasesthe affinity of the C_(H)3 amino acid sequence to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,N434S, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,V259I, V308F, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising an N434A mutation.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M252Y,S254T, T256E, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of T250Q,M248L, or both.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of H433K,N434Y, or both.

In one embodiment, the genetically modified immunoglobulin locuscomprises: (1) a first allele, wherein the unrearranged humanimmunoglobulin heavy chain variable region nucleotide sequence asdescribed herein is operably linked to a first heavy chain constantregion nucleotide sequence encoding a first CH₃ amino acid sequence of ahuman IgG selected from IgG1, IgG2, IgG4, and a combination thereof; and(2) a second allele, wherein the unrearranged human immunoglobulin heavychain variable region nucleotide sequence as described herein isoperably linked to a second heavy chain constant region nucleotidesequence encoding a second C_(H)3 amino acid sequence of the human IgGselected from IgG1, IgG2, IgG4, and a combination thereof, and whereinthe second CH₃ amino acid sequence comprises a modification that reducesor eliminates binding for the second CH₃ amino acid sequence to ProteinA (see, for example, US 2010/0331527A1, which is incorporated byreference herein in its entirety).

In one embodiment, the second CH₃ amino acid sequence comprises an H95Rmodification (by IMGT exon numbering; H435R by EU numbering). In oneembodiment the second CH₃ amino acid sequence further comprises an Y96Fmodification (by IMGT exon numbering; H436F by EU). In anotherembodiment, the second CH₃ amino acid sequence comprises both an H95Rmodification (by IMGT exon numbering; H435R by EU numbering) and an Y96Fmodification (by IMGT exon numbering; H436F by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG1 and further comprises a mutation selected from the groupconsisting of D16E, L18M, N44S, K52N, V57M, and V82I (IMGT; D356E, L38M,N384S, K392N, V397M, and V422I by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG2 and further comprises a mutation selected from the groupconsisting of N44S, K52N, and V82I (IMGT: N384S, K392N, and V422I byEU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG4 and further comprises a mutation selected from the groupconsisting of Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (IMGT: Q355R,N384S, K392N, V397M, R409K, E419Q, and V422I by EU).

In one embodiment, the heavy chain constant region amino acid sequenceis a non-human constant region amino acid sequence, and the heavy chainconstant region amino acid sequence comprises one or more of any of thetypes of modifications described above.

In one embodiment, the heavy chain constant region nucleotide sequenceis a human heavy chain constant region amino acid sequence, and thehuman heavy chain constant region amino acid sequence comprises one ormore of any of the types of modifications described above.

In one embodiment, all or substantially all endogenous V_(H), D, andJ_(H) gene segments are deleted from an immunoglobulin heavy chain locusor rendered non-functional (e.g., via insertion of a nucleotide sequence(e.g., an exogenous nucleotide sequence) in the immunoglobulin locus orvia non-functional rearrangement, or inversion, of the endogenous V_(H),D, J_(H) segments). In one embodiment, e.g., about 80% or more, about85% or more, about 90% or more, about 95% or more, about 96% or more,about 97% or more, about 98% or more, or about 99% or more of allendogenous V_(H), D, or J_(H) gene segments are deleted or renderednon-functional. In one embodiment, e.g., at least 95%, 96%, 97%, 98%, or99% of endogenous functional V, D, or J gene segments are deleted orrendered non-functional.

In one embodiment, the genetically modified immunoglobulin heavy chainlocus comprises a modification that deletes or renders, all orsubstantially all, non-functional endogenous V_(H), D, and J_(H) genesegments; and the genetically modified locus comprises an unrearrangedheavy chain variable region nucleotide sequence comprising one or morehuman V_(H), D, and/or J_(H) gene segments having one or more histidinecodons, wherein the unrearranged heavy chain variable region nucleotidesequence is present at an endogenous location (i.e., where thenucleotide sequence is located in a wild-type non-human animal) orpresent ectopically (e.g., at a locus different from the endogenousimmunoglobulin chain locus in its genome, or within its endogenouslocus, e.g., within an immunoglobulin variable locus, wherein theendogenous locus is placed or moved to a different location in thegenome).

In one embodiment, the genetically modified immunoglobulin locuscomprises an endogenous Adam6a gene, Adam6b gene, or both, and thegenetic modification does not affect the expression and/or function ofthe endogenous Adam6a gene, Adam6b gene, or both.

In one embodiment, the genetically modified immunoglobulin locuscomprises an ectopically present Adam6a gene, Adam6b gene, or both. Inone embodiment, the Adam6a gene is a non-human Adam6a gene. In oneembodiment, the Adam6a gene is a human Adam6a gene. In one embodiment,the Adam6b gene is a non-human Adam6b gene. In one embodiment, theAdam6b gene is a human Adam6b gene.

In one embodiment, the genetically modified immunoglobulin locus furthercomprises a humanized, unrearranged λ and/or κ light chain variable genesequence. In one embodiment, the humanized, unrearranged λ and/or κlight chain variable gene sequence is operably linked to animmunoglobulin light chain constant region nucleotide sequence selectedfrom a λ light chain constant region nucleotide sequence and a κ lightchain constant region nucleotide sequence. In one embodiment, thehumanized, unrearranged λ light chain variable region nucleotidesequence is operably linked to a λ light chain constant regionnucleotide sequence. In one embodiment, the λ light chain constantregion nucleotide sequence is a mouse, rat, or human sequence. In oneembodiment, the humanized, unrearranged κ light chain variable regionnucleotide sequence is operably linked to a κ light chain constantregion nucleotide sequence. In one embodiment, the κ light chainconstant region nucleotide sequence is a mouse, rat, or human sequence.

In one embodiment, the genetically modified immunoglobulin locuscomprises an unrearranged light chain variable gene sequence thatcontains at least one modification that introduces at least onehistidine codon in at least one reading frame encoding a light chainvariable domain. In one embodiment, the genetically modifiedimmunoglobulin locus comprises a rearranged (e.g., a rearranged λ or κV/J sequence) sequence that comprises one, two, three, or four codonsfor histidine in a light chain CDR. In one embodiment, the CDR is aselected from a CDR1, CDR2, CDR3, and a combination thereof. In oneembodiment, the unrearranged or rearranged light chain variable regionnucleotide sequence is an unrearranged or rearranged human λ or κ lightchain variable region nucleotide sequence. In one embodiment, theunrearranged or rearranged human λ or κ light chain variable regionnucleotide sequence is present at an endogenous mouse immunoglobulinlight chain locus. In one embodiment, the mouse immunoglobulin lightchain locus is a mouse κ locus. In one embodiment, the mouseimmunoglobulin light chain locus is a mouse λ locus.

In one embodiment, the genetically modified immunoglobulin locus asdescribed herein is present in an immunoglobulin heavy chain locus of amouse. In one embodiment, the genetically modified immunoglobulin locusis present in a humanized immunoglobulin heavy chain locus in aVELOCIMMUNE® mouse.

In one embodiment, the non-human animal is heterozygous for thegenetically modified immunoglobulin heavy chain locus, and the non-humananimal is capable of expressing a human immunoglobulin heavy chainvariable domain comprising at least one histidine residue derivedpredominantly from the genetically modified immunoglobulin heavy chainlocus as described herein.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein exhibits a weaker antigen bindingat an acidic environment (e.g., at a pH of about 5.5 to about 6.0) thana corresponding wild-type heavy chain variable domain without thegenetic modification.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 2 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 25° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin heavy chain locus as described herein has adissociative half-life (t_(1/2)) of less than 2 min at an acidic pH(e.g., pH of about 5.5 to about 6.0) at 37° C. In one embodiment, anantigen-binding protein comprising a heavy chain variable domainexpressed by the genetically modified immunoglobulin heavy chain locusas described herein has a dissociative half-life (t_(1/2)) of less than1 min at an acidic pH (e.g., pH of about 5.5 to about 6.0) at 25° C. Inone embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 1 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 37° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin locus as described herein has at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold, at least about 25-fold, or at least about 30-fold decrease indissociative half-life (t_(1/2)) at an acidic pH (e.g., pH of about 5.5to about 6.0) as compared to the dissociative half-life (t_(1/2)) of theantigen-binding protein at a neutral pH (e.g., pH of about 7.0 to about7.4).

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinlocus as described herein is characterized by improved pH-dependentrecyclability, enhanced serum half-life, or both as compared with awild-type antigen-binding protein without the genetic modification.

In one embodiment, the genetically modified immunoglobulin locusdescribed herein comprises a B cell population that, upon stimulationwith an antigen of interest, is capable of producing antigen-bindingproteins, e.g., antibodies, comprising a heavy chain variable domaincomprising one or more histidine residues. The antigen-binding proteinsas described herein when administered into a subject, exhibits anincreased serum half-life over a corresponding wild-type antigen-bindingprotein, which possesses a similar or sufficiently similar amino acidsequence that encodes the heavy chain variable domain but does notcomprise a histidine residue in the heavy chain variable domain. In someembodiments, the antigen-binding protein described herein exhibits anincreased serum half-life that is at least about 2-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold higher than the corresponding wild-type antigen-binding protein,which possesses a similar or sufficiently similar amino acid sequencethat encodes the heavy chain variable domain but does not comprise ahistidine residue in the heavy chain variable domain.

In one aspect, a non-human animal comprising a genetically modifiedimmunoglobulin locus is provided, wherein the genetically modifiedimmunoglobulin locus comprises an unrearranged human heavy chainvariable region nucleotide sequence, and wherein the human unrearrangedheavy chain variable region nucleotide sequence comprises a substitutionof at least one endogenous non-histidine codon with a histidine codon.

In one embodiment, the non-human animal is a mammal, including a rodent,e.g., a mouse, a rat, or a hamster.

In one embodiment, 2 or more, 3 or more, 4 or more, 5 or more, 6 ormore, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 ormore, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 ormore, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 ormore, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 ormore, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 ormore, 37 or more, 38 or more, 39 or more, 40 or more, 41 or more, 42 ormore, 43 or more, 44 or more, 45 or more, 46 or more, 47 or more, 48 ormore, 49 or more, 50 or more, 51 or more, 52 or more, 53 or more, 54 ormore, 55 or more, 56 or more, 57 or more, 58 or more, 59 or more, 60 ormore, or 61 or more of the endogenous non-histidine codons are replacedwith histidine codons.

In one embodiment, the endogenous non-histone codon encodes the aminoacid selected from Y, N, D, Q, S, W, and R.

In one embodiment, the substituted histidine codon is present in anunrearranged heavy chain variable region nucleotide sequence thatencodes an immunoglobulin variable domain selected from an N-terminalregion, a loop 4 region, a CDR1, a CDR2, a CDR3, a combination thereof.

In one embodiment, the substituted histidine codon is present in anunrearranged heavy chain variable region nucleotide sequence thatencodes a complementary determining region (CDR) selected from a CDR1, aCDR2, a CDR3, and a combination thereof.

In one embodiment, the substituted histidine codon is present in anunrearranged heavy chain variable region nucleotide sequence thatencodes a frame region (FR) selected from FR1, FR2, FR3, FR4, and acombination thereof.

In one embodiment, the unrearranged heavy chain variable regionnucleotide sequence comprises a genetically modified human V_(H) genesegment, wherein one or more endogenous non-histidine codon in at leastone reading frame of the human V_(H) gene segment has been replaced witha histidine codon.

In one embodiment, the human unrearranged heavy chain variable regionnucleotide sequence comprises a modification that replaces at least oneendogenous non-histidine codon of a human V_(H) gene segment with ahistidine codon, wherein the human V_(H) gene segment is selected fromthe group consisting of V_(H)1-2, V_(H)1-3, V_(H)1-8, V_(H)1-18,V_(H)1-24, V_(H)1-45, V_(H)1-46, V_(H)1-58, V_(H)1-69, V_(H)2-5,V_(H)2-26, V_(H)2-70, V_(H)3-7, V_(H)3-9, V_(H)3-11, V_(H)3-13,V_(H)3-15, V_(H)3-16, V_(H)3-20, V_(H)3-21, V_(H)3-23, V_(H)3-30,V_(H)3-30-3, V_(H) 3-30-5, V_(H)3-33, V_(H)3-35, V_(H)3-38, V_(H)3-43,V_(H)3-48, V_(H)3-49, V_(H)3-53, V_(H)3-64, V_(H)3-66, V_(H)3-72,V_(H)3-73, V_(H)3-74, V_(H)4-4, V_(H)4-28, V_(H)4-30-1, V_(H)4-30-2,V_(H)4-30-4, V_(H)4-31, V_(H)4-34, V_(H)4-39, V_(H)4-59, V_(H)4-61,V_(H)5-51, V_(H)6-1, V_(H)7-4-1, V_(H)7-81, and a combination thereof.

In one embodiment, the human unrearranged heavy chain variable regionnucleotide sequence comprises a genetically modified human J_(H) genesegment, wherein one or more endogenous non-histidine codon in at leastone reading frame of the human J_(H) gene segment has been replaced witha histidine codon.

In one embodiment, the human unrearranged heavy chain variable regionnucleotide sequence comprises a modification that replaces at least oneendogenous non-histidine codon of a human J_(H) segment with a histidinecodon, wherein the human J_(H) gene segment is selected from the groupconsisting of J_(H)1, J_(H)2, J_(H)3, J_(H)4, J_(H)5, J_(H)6, and acombination thereof.

In one embodiment, the substituted histidine codon is present in a heavychain variable region nucleotide sequence that encodes part of a CDR3.In one embodiment, the part of CDR3 comprises an amino acid sequencederived from a reading frame of a genetically modified human D genesegment comprising a modification that replaces at least one endogenousnon-histidine codon in the reading frame with a histidine codon.

In one embodiment, the endogenous non-histidine codon that issubstituted with a histidine codon encodes the amino acid selected fromY, N, D, Q, S, W, and R.

In one embodiment, the substituted histidine codon is present in atleast one reading frame of the human D gene segment that is mostfrequently observed in VELOCIMMUNE® humanized immunoglobulin mice.

In one embodiment, the reading frame of the genetically modified human Dgene segment that encodes part of CDR3 is selected from a hydrophobicframe, a stop frame, and a hydrophilic frame.

In one embodiment, the reading frame is a hydrophobic frame of a human Dgene segment.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D1-1 (GTTGT; SEQ ID NO: 88), D1-7(GITGT; SEQ ID NO: 89), D1-20 (GITGT; SEQ ID NO: 89), and D1-26 (GIVGAT;SEQ ID NO:90), and the human D gene segment further comprises amodification that replaces at least one endogenous non-histidine codonin the nucleotide sequence with a histidine codon.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D2-2 (DIVVVPAAI; SEQ ID NO:92),D2-8 (DIVLMVYAI; SEQ ID NO: 94), D2-15 (DIVVVVAAT; SEQ ID NO:95), andD2-21 (HIVVVTAI; SEQ ID NO: 97), and the human D gene segment furthercomprises a modification that replaces at least one endogenousnon-histidine codon in the nucleotide sequence with a histidine codon.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D3-3 (ITIFGVVII; SEQ ID NO:98),D3-9 (ITIF*LVII; SEQ ID NO:99, SEQ ID NO:100), D3-10 (ITMVRGVII; SEQ IDNO:101), D3-16 (IMITFGGVIVI; SEQ ID NO:102), and D3-22 (ITMIVVVIT; SEQID NO:103), and the human D gene segment further comprises amodification that replaces at least one endogenous codon in thenucleotide sequence with a histidine codon.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D4-4 (TTVT; SEQ ID NO:105), D4-11(TTVT; SEQ ID NO:105), D4-17 (TTVT; SEQ ID NO:105), D4-23 (TTVVT; SEQ IDNO: 106) and the human D gene segment further comprises a modificationthat replaces at least one endogenous non-histidine codon in thenucleotide sequence with a histidine codon.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D5-5 (VDTAMV; SEQ ID NO: 107),D5-12 (VDIVATI; SEQ ID NO:108), D5-18 (VDTAMV; SEQ ID NO:107), and D5-24(VEMATI; SEQ ID NO:109), and the human D gene segment further comprisesa modification that replaces at least one endogenous non-histidine codonin the nucleotide sequence with a histidine codon.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D6-6 (SIAAR; SEQ ID NO:111), D6-13(GIAAAG; SEQ ID NO:113), and D6-19 (GIAVAG; SEQ ID NO:115), and thehuman D gene segment further comprises a modification that replaces atleast one endogenous non-histidine codon in the nucleotide sequence witha histidine codon.

In one embodiment, the hydrophobic frame comprises a nucleotide sequencethat encodes human D7-27 (LTG), and the human D gene segment furthercomprises a modification that replaces at least one endogenousnon-histidine codon in the nucleotide sequence with a histidine codon.

In one embodiment, the reading frame is a stop reading frame of a humanD gene segment.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D1-1 (VQLER; SEQ ID NO:8),D1-7(V*LEL), D1-20(V*LER), D1-26 (V*WELL; SEQ ID NO:12), and the human Dgene segment further comprises a modification that replaces at least oneendogenous non-histidine codon in the nucleotide sequence with ahistidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D2-2 (RIL**YQLLY; SEQ ID NO:14),D2-8 (RILY*WCMLY; SEQ ID NO:16 and SEQ ID NO: 17), D2-15 (RIL*WW*LLL),and D2-21 (SILWW*LLF; SEQ ID NO:19), and the human D gene segmentfurther comprises a modification that replaces at least one endogenousnon-histidine codon in the nucleotide sequence with a histidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D3-3 (VLRFLEWLLY; SEQ ID NO:21),D3-9 (VLRYFDWLL*; SEQ ID NO:23), D3-10 (VLLWFGELL*; SEQ ID NO:25), D3-16(VL*LRLGELSLY; SEQ ID NO:27), and D3-22 (VLL***WLLL; SEQ ID NO:29), andthe human D gene segment comprises a modification that replaces at leastone endogenous non-histidine codon in the nucleotide sequence with ahistidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D4-4 (*LQ*L), D4-11 (*LQ*L), D4-17(*LR*L), and D4-23 (*LRW*L), and the human D gene segment comprises amodification that replaces at least one endogenous non-histidine codonin the nucleotide sequence with a histidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D5-5 (WIQLWL; SEQ ID NO:35); D5-12(Wl*WLRL; SEQ ID NO:37), D5-18 (WIQLWL; SEQ ID NO:35), and D5-24(*RWLQL; SEQ ID NO:39), and the human D gene segment comprises amodification that replaces at least one endogenous non-histidine codonin the nucleotide sequence with a histidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D6-6 (V*QLV), D6-13 (V*QQLV; SEQID NO:41), and D6-19 (V*QWLV; SEQ ID NO:43), and the human D genesegment further comprises a modification that replaces at least oneendogenous non-histidine codon in the nucleotide sequence with ahistidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes D7-27 (*LG), and the humanD gene segment further comprises a modification that replaces at leastone endogenous codon of the human D gene segment in the nucleotidesequence with a histidine codon.

In one embodiment, the reading frame is a hydrophilic frame of a human Dgene segment.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D1-1 (YNWND; SEQ ID NO: 45), D1-7(YNWNY; SEQ ID NO: 47), D1-20 (YNWND; SEQ ID NO: 45), and D1-26 (YSGSYY;SEQ ID NO:49), and the human D gene segment further comprises amodification that replaces at least one endogenous codon in thenucleotide sequence with a histidine codon. In one embodiment, thehydrophilic frame comprises a nucleotide sequence that encodes the aminoacid sequence selected from the group consisting of SEQ ID NO: 46, SEQID NO: 48, SEQ ID NO: 50, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D2-2 (GYCSSTSCYT; SEQ ID NO:51),D2-8 (GYCTNGVCYT; SEQ ID NO: 53), D2-15 (GYCSGGSCYS; SEQ ID NO:55), andD2-21 (AYCGGDCYS; SEQ ID NO:57), and the human D gene segment furthercomprises a modification that replaces at least one endogenous codon inthe nucleotide sequence with a histidine codon. In one embodiment, thehydrophilic frame comprises a nucleotide sequence that encodes the aminoacid sequence selected from the group consisting of SEQ ID NO: 52, SEQID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D3-3 (YYDFWSGYYT; SEQ ID NO:59),D3-9 (YYDILTGYYN; SEQ ID NO:61), D3-10 (YYYGSGSYYN; SEQ ID NO:63), D3-16(YYDYVWGSYRYT; SEQ ID NO:65), and D3-22 (YYYDSSGYYY; SEQ ID NO:67), andthe human D gene segment further comprises a modification that replacesat least one endogenous codon in the nucleotide sequence with ahistidine codon. In one embodiment, the hydrophilic frame comprises anucleotide sequence encodes the amino acid sequence selected from thegroup consisting of SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ IDNO: 66, SEQ ID NO: 68, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D4-4 (DYSNY; SEQ ID NO:69), D4-11(DYSNY; SEQ ID NO:69), D4-17 (DYGDY; SEQ ID NO:71), and D4-23 (DYGGNS;SEQ ID NO:73), and the human D gene segment comprises a modificationthat replaces at least one endogenous codon in the nucleotide sequencewith a histidine codon. In one embodiment, the hydrophilic framecomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of SEQ ID NO: 70, SEQ ID NO: 72, SEQID NO: 74, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D5-5 (GYSYGY; SEQ ID NO:75), D5-12(GYSGYDY; SEQ ID NO:77), D5-18 (GYSYGY; SEQ ID NO:75), and D5-24(RDGYNY; SEQ ID NO:79), and the human D gene segment further comprises amodification that replaces at least one endogenous codon in thenucleotide sequence with a histidine codon. In one embodiment, thehydrophilic frame comprises a nucleotide sequence that encodes the aminoacid sequence selected from the group consisting of SEQ ID NO: 76, SEQID NO: 78, SEQ ID NO: 80, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D6-6 (EYSSSS; SEQ ID NO: 81),D6-13 (GYSSSWY; SEQ ID NO:83), and D6-19 (GYSSGWY; SEQ ID NO:85), andthe human D gene segment further comprises a modification that replacesat least one endogenous codon in the nucleotide sequence with ahistidine codon. In one embodiment, the hydrophilic frame comprises anucleotide sequence that encodes the amino acid sequence selected fromthe group consisting of SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQID NO: 76, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes D7-27 (NWG), and the humanD gene segment further comprises a modification that replaces at leastone endogenous codon in the nucleotide sequence a histidine codon.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 48, SEQID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO:68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ IDNO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, anda combination thereof.

In one embodiment, the unrearranged heavy chain variable regionnucleotide sequence comprising the inverted human D gene segment isoperably linked to a human or non-human heavy chain constant regionnucleotide sequence that encodes an immunoglobulin isotype selected fromIgM, IgD, IgG, IgE, and IgA.

In one embodiment, the human unrearranged immunoglobulin heavy chainvariable region nucleotide sequence is operably linked to a human ornon-human heavy chain constant region nucleotide sequence selected froma C_(H)1, a hinge, a C_(H)2, a C_(H)3, and a combination thereof. In oneembodiment, the heavy chain constant region nucleotide sequencecomprises a C_(H)1, a hinge, a C_(H)2, and a C_(H)3 (i.e.,C_(H)1-hinge-C_(H)2-C_(H)3).

In one embodiment, a heavy chain constant region nucleotide sequence ispresent at an endogenous locus (i.e., where the nucleotide sequence islocated in a wild-type non-human animal) or present ectopically (e.g.,at a locus different from the endogenous immunoglobulin chain locus inits genome, or within its endogenous locus, e.g., within animmunoglobulin variable locus, wherein the endogenous locus is placed ormoved to a different location in the genome).

In one embodiment, the heavy chain constant region nucleotide sequencecomprises a modification in a C_(H)2 or a C_(H)3, wherein themodification increases the affinity of the heavy chain constant regionamino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a modification at position 250 (e.g., E or Q); 250 and 428(e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256(e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433(e.g., L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification atposition 250 and/or 428; or a modification at position 307 or 308 (e.g.,308F, V308F), and 434. In one embodiment, the modification comprises a428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I(e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K)and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y,254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Qand M428L); and a 307 and/or 308 modification (e.g., 308F or 308P),wherein the modification increases the affinity of the heavy chainconstant region amino acid sequence to FcRn in an acidic environment(e.g., in an endosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 252 and 257,wherein the modification increases the affinity of the human C_(H)2amino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 307 and 311,wherein the modification increases the affinity of the C_(H)2 amino acidsequence to FcRn in an acidic environment (e.g., in an endosome where pHranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)3 amino acid sequence, wherein the C_(H)3 aminoacid sequence comprises at least one modification between amino acidresidues at positions 433 and 436, wherein the modification increasesthe affinity of the C_(H)3 amino acid sequence to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,N434S, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,V259I, V308F, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising an N434A mutation.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M252Y,S254T, T256E, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of T250Q,M248L, or both.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of H433K,N434Y, or both.

In one embodiment, the genetically modified immunoglobulin locuscomprises: (1) a first allele, wherein the unrearranged humanimmunoglobulin heavy chain variable region nucleotide sequence asdescribed herein is operably linked to a first heavy chain constantregion nucleotide sequence encoding a first CH₃ amino acid sequence of ahuman IgG selected from IgG1, IgG2, IgG4, and a combination thereof; and(2) a second allele, wherein the unrearranged human immunoglobulin heavychain variable region nucleotide sequence as described herein isoperably linked to a second heavy chain constant region nucleotidesequence encoding a second C_(H)3 amino acid sequence of the human IgGselected from IgG1, IgG2, IgG4, and a combination thereof, and whereinthe second CH₃ amino acid sequence comprises a modification that reducesor eliminates binding for the second CH₃ amino acid sequence to ProteinA (see, for example, US 2010/0331527A1, which is incorporated byreference herein in its entirety).

In one embodiment, the second CH₃ amino acid sequence comprises an H95Rmodification (by IMGT exon numbering; H435R by EU numbering). In oneembodiment the second CH₃ amino acid sequence further comprises an Y96Fmodification (by IMGT exon numbering; H436F by EU). In anotherembodiment, the second CH₃ amino acid sequence comprises both an H95Rmodification (by IMGT exon numbering; H435R by EU numbering) and an Y96Fmodification (by IMGT exon numbering; H436F by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG1 and further comprises a mutation selected from the groupconsisting of D16E, L18M, N44S, K52N, V57M, and V82I (IMGT; D356E, L38M,N384S, K392N, V397M, and V422I by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG2 and further comprises a mutation selected from the groupconsisting of N44S, K52N, and V82I (IMGT: N384S, K392N, and V422I byEU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG4 and further comprises a mutation selected from the groupconsisting of Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (IMGT: Q355R,N384S, K392N, V397M, R409K, E419Q, and V422I by EU).

In one embodiment, the heavy chain constant region amino acid sequenceis a non-human constant region amino acid sequence, and the heavy chainconstant region amino acid sequence comprises one or more of any of thetypes of modifications described above.

In one embodiment, the heavy chain constant region nucleotide sequenceis a human heavy chain constant region amino acid sequence, and thehuman heavy chain constant region amino acid sequence comprises one ormore of any of the types of modifications described above.

In one embodiment, all, or substantially all, endogenous V_(H), D, andJ_(H) gene segments are deleted from an immunoglobulin heavy chain locusor rendered non-functional (e.g., via insertion of a nucleotide sequence(e.g., an exogenous nucleotide sequence) in the immunoglobulin locus orvia non-functional rearrangement, or inversion, of the endogenous V_(H),D, J_(H) segments). In one embodiment, e.g., about 80% or more, about85% or more, about 90% or more, about 95% or more, about 96% or more,about 97% or more, about 98% or more, or about 99% or more of allendogenous V_(H), D, or J_(H) gene segments are deleted or renderednon-functional. In one embodiment, e.g., at least 95%, 96%, 97%, 98%, or99% of endogenous functional V, D, or J gene segments are deleted orrendered non-functional.

In one embodiment, the genetically modified locus comprises amodification that deletes or renders non-functional all or substantiallyall endogenous V_(H), D, and J_(H) gene segments; and the genomic locuscomprises the genetically modified, unrearranged human heavy chainvariable region nucleotide sequence comprising a substitution of atleast one endogenous non-histidine codon with a histidine codon in atleast one reading frame. In one embodiment, the genetically modified,unrearranged immunoglobulin heavy chain variable gene sequence ispresent at an endogenous location (i.e., where the nucleotide sequenceis located in a wild-type non-human animal) or present ectopically(e.g., at a locus different from the endogenous immunoglobulin chainlocus in its genome), or within its endogenous locus, e.g., within animmunoglobulin variable locus, wherein the endogenous locus is placed ormoved to a different location in the genome.

In one embodiment, the genetically modified locus comprises anendogenous Adam6a gene, Adam6b gene, or both, and the geneticmodification does not affect the expression and/or function of theendogenous Adam6a gene, Adam6b gene, or both.

In one embodiment, the genetically modified locus comprises anectopically present Adam6a gene, Adam6b gene, or both. In oneembodiment, the Adam6a gene is a non-human Adam6a gene. In oneembodiment, the Adam6a gene is a mouse Adam6a gene. In one embodiment,the Adam6a gene is a human Adam6a gene. In one embodiment, the Adam6bgene is a non-human Adam6b gene. In one embodiment, the Adam6b gene is amouse Adam6b gene. In one embodiment, the Adam6b gene is a human Adam6bgene.

In one embodiment, the genetically modified immunoglobulin locus furthercomprises a humanized, unrearranged λ and/or κ light chain variable genesequence. In one embodiment, the humanized, unrearranged λ and/or κlight chain variable gene sequence is operably linked to animmunoglobulin light chain constant region nucleotide sequence selectedfrom a λ light chain constant region nucleotide sequence and a κ lightchain constant region nucleotide sequence. In one embodiment, thehumanized, unrearranged λ light chain variable region nucleotidesequence is operably linked to a λ light chain constant regionnucleotide sequence. In one embodiment, the λ light chain constantregion nucleotide sequence is a mouse, rat, or human sequence. In oneembodiment, the humanized, unrearranged κ light chain variable regionnucleotide sequence is operably linked to a κ light chain constantregion nucleotide sequence. In one embodiment, the κ light chainconstant region nucleotide sequence is a mouse, rat, or human sequence.

In one embodiment, the genetically modified immunoglobulin locuscomprises an unrearranged light chain variable gene sequence thatcontains at least one modification that introduces at least onehistidine codon in at least one reading frame encoding a light chainvariable domain. In one embodiment, the genetically modifiedimmunoglobulin locus comprises a rearranged (e.g., rearranged λ or κ V/Jsequence) sequence that comprises one, two, three, or four codons forhistidine in a light chain CDR. In one embodiment, the CDR is a selectedfrom a CDR1, CDR2, CDR3, and a combination thereof. In one embodiment,the unrearranged or rearranged light chain variable region nucleotidesequence is an unrearranged or rearranged human λ or κ light chainvariable region nucleotide sequence. In one embodiment, the unrearrangedor rearranged human λ or κ light chain variable region nucleotidesequence is present at an endogenous mouse immunoglobulin light chainlocus. In one embodiment, the mouse immunoglobulin light chain locus isa mouse κ locus. In one embodiment the mouse immunoglobulin light chainlocus is a mouse λ locus.

In one embodiment, the genetically modified immunoglobulin locus asdescribed herein is present in an immunoglobulin heavy chain locus of amouse. In one embodiment, the genetically modified immunoglobulin locusis present in a humanized immunoglobulin heavy chain locus in aVELOCIMMUNE® mouse.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein exhibits a weaker antigen bindingat an acidic environment (e.g., at a pH of about 5.5 to about 6.0) thana corresponding wild-type heavy chain variable domain without thegenetic modification.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 2 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 25° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin heavy chain locus as described herein has adissociative half-life (t_(1/2)) of less than 2 min at an acidic pH(e.g., pH of about 5.5 to about 6.0) at 37° C. In one embodiment, anantigen-binding protein comprising a heavy chain variable domainexpressed by the genetically modified immunoglobulin heavy chain locusas described herein has a dissociative half-life (t_(1/2)) of less than1 min at an acidic pH (e.g., pH of about 5.5 to about 6.0) at 25° C. Inone embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 1 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 37° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin locus as described herein has at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold, at least about 25-fold, or at least about 30-fold decrease indissociative half-life (t_(1/2)) at an acidic pH (e.g., pH of about 5.5to about 6.0) as compared to the dissociative half-life (t_(1/2)) of theantigen-binding protein at a neutral pH (e.g., pH of about 7.0 to about7.4).

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinlocus as described herein is characterized by improved pH-dependentrecyclability, enhanced serum half-life, or both as compared with awild-type antigen-binding protein without the genetic modification.

In one embodiment, the genetically modified immunoglobulin locus asdescribed herein comprises a B cell population that, upon stimulationwith an antigen of interest, is capable of producing antigen-bindingproteins, e.g., antibodies, comprising a heavy chain variable domaincomprising one or more histidine residues. The antigen-binding proteinsas described herein, when administered into a subject, exhibits anincreased serum half-life over a corresponding wild-type antigen-bindingprotein, which possesses a similar or sufficiently similar amino acidsequence that encodes the heavy chain variable domain but does notcomprise a histidine residue in the heavy chain variable domain. In someembodiments, the antigen-binding protein described herein exhibits anincreased serum half-life that is at least about 2-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold higher than the corresponding wild-type antigen-binding protein,which possesses a similar or sufficiently similar amino acid sequencethat encodes the heavy chain variable domain but does not comprise ahistidine residue in the heavy chain variable domain.

In one embodiment, the non-human animal is heterozygous for thegenetically modified immunoglobulin heavy chain locus, and the non-humananimal is capable of expressing the human immunoglobulin heavy chainvariable domain comprising at least one histidine residue derivedpredominantly from the genetically modified immunoglobulin heavy chainlocus as described herein.

In one aspect, a non-human animal comprising a genetically modifiedimmunoglobulin locus comprising a human V_(H), D, and J_(H) gene segmentis provided, wherein at least one of the human D gene segment has beeninverted 5′ to 3′ with respect to a corresponding wild-type sequence,and wherein at least one reading frame of the inverted human D genesegment comprises a histidine codon.

In one embodiment, the non-human animal is a mammal, including a rodent,e.g., a mouse, a rat, or a hamster

In one embodiment, the genetically modified immunoglobulin locus ispresent in a germline genome.

In one embodiment, wherein the reading frame of the inverted human Dgene segment comprises one or more, 2 or more, 3 or more, 4 or more, 5or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 ormore, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 ormore, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 ormore, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 ormore, 30 or more, 31 or more, 32 or more, 33 or more, or 34 or more ofhistidine codons.

In one embodiment, at least two, at least three, at least four, at leastfive, at least six, at least seven, at least eight, at least nine, atleast ten, at least eleven, at least twelve, at least thirteen, at leastfourteen, at least fifteen, at least sixteen, at least seventeen, atleast eighteen, at least nineteen, at least twenty, at least twenty one,at least twenty two, at least twenty three, at least twenty four, or allor substantially all of functional human D gene segments have invertedorientation with respect to corresponding wild type sequences.

In one embodiment, all or substantially all of endogenous immunoglobulinV_(H), D, J_(H) gene segments are deleted from the immunoglobulin heavychain locus or rendered non-functional (e.g., via insertion of anucleotide sequence, e.g., exogenous nucleotide sequence, in theimmunoglobulin locus or via non-functional rearrangement or inversion ofall, or substantially all, endogenous immunoglobulin V_(H), D, J_(H)segments), and the genetically modified immunoglobulin locus comprises ahuman V_(H), D, and J_(H) gene segments, wherein at least one of thehuman D gene segment is present in an inverted orientation with respectto corresponding wild type sequences, and wherein at least one readingframe of the inverted human D gene segment comprises at least onehistidine codon.

In one embodiment, the inverted human D gene segment is operably linkedto a human V_(H) gene segment, and/or human J_(H) gene segment

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence isselected from the group consisting of D1-1, D1-7, D1-20, D1-26, D2-2,D2-8, D2-15, D2-21, D3-3, D3-9, D3-10, D3-16, D3-22, D4-4, D4-11, D4-17,D4-23, D5-5, D5-12, D5-18, D5-24, D6-6, D6-13, D6-19, D7-27, and acombination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD1 gene segment selected from the group consisting of D1-1, D1-7, D1-20,D1-26, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequences isa D2 gene segment selected from the group consisting of D2-2, D2-8,D2-15, D2-21, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD3 gene segment selected from the group consisting of D3-3, D3-9, D3-10,D3-16, D3-22, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD4 gene segment selected from the group consisting of D4-4, D4-11,D4-17, D4-23, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD5 gene segment selected from the group consisting of D5-5, D5-12,D5-18, D5-24, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD6 gene segment selected from the group consisting of D6-6, D6-13,D6-19, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence isD7-27.

In one embodiment, the reading frame of the human D gene segment isselected from a stop reading frame, a hydrophilic reading frame, ahydrophobic reading frame, and a combination thereof, wherein at leastone reading frame of the inverted human D gene segment comprises ahistidine codon.

In one embodiment, the unrearranged heavy chain variable regionnucleotide sequence comprising the inverted human D gene segment isoperably linked to a human or non-human heavy chain constant regionnucleotide sequence that encodes an immunoglobulin isotype selected fromIgM, IgD, IgG, IgE, and IgA.

In one embodiment, the human unrearranged immunoglobulin heavy chainvariable region nucleotide sequence is operably linked to a human ornon-human heavy chain constant region nucleotide sequence selected froma C_(H)1, a hinge, a C_(H)2, a C_(H)3, and a combination thereof. In oneembodiment, the heavy chain constant region nucleotide sequencecomprises a C_(H)1, a hinge, a C_(H)2, and a C_(H)3 (i.e.,C_(H)1-hinge-C_(H)2-C_(H)3).

In one embodiment, a heavy chain constant region nucleotide sequence ispresent at an endogenous locus (i.e., where the nucleotide sequence islocated in a wild-type non-human animal) or present ectopically (e.g.,at a locus different from the endogenous immunoglobulin chain locus inits genome, or within its endogenous locus, e.g., within animmunoglobulin variable locus, wherein the endogenous locus is placed ormoved to a different location in the genome).

In one embodiment, the heavy chain constant region nucleotide sequencecomprises a modification in a C_(H)2 or a C_(H)3, wherein themodification increases the affinity of the heavy chain constant regionamino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a modification at position 250 (e.g., E or Q); 250 and 428(e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256(e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433(e.g., L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification atposition 250 and/or 428; or a modification at position 307 or 308 (e.g.,308F, V308F), and 434. In one embodiment, the modification comprises a428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I(e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K)and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y,254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Qand M428L); and a 307 and/or 308 modification (e.g., 308F or 308P),wherein the modification increases the affinity of the heavy chainconstant region amino acid sequence to FcRn in an acidic environment(e.g., in an endosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 252 and 257,wherein the modification increases the affinity of the human C_(H)2amino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 307 and 311,wherein the modification increases the affinity of the C_(H)2 amino acidsequence to FcRn in an acidic environment (e.g., in an endosome where pHranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)3 amino acid sequence, wherein the C_(H)3 aminoacid sequence comprises at least one modification between amino acidresidues at positions 433 and 436, wherein the modification increasesthe affinity of the C_(H)3 amino acid sequence to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,N434S, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,V259I, V308F, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising an N434A mutation.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M252Y,S254T, T256E, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of T250Q,M248L, or both.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of H433K,N434Y, or both.

In one embodiment, the genetically modified immunoglobulin locuscomprises: (1) a first allele, wherein the unrearranged humanimmunoglobulin heavy chain variable region nucleotide sequence asdescribed herein is operably linked to a first heavy chain constantregion nucleotide sequence encoding a first CH₃ amino acid sequence of ahuman IgG selected from IgG1, IgG2, IgG4, and a combination thereof; and(2) a second allele, wherein the unrearranged human immunoglobulin heavychain variable region nucleotide sequence as described herein isoperably linked to a second heavy chain constant region nucleotidesequence encoding a second C_(H)3 amino acid sequence of the human IgGselected from IgG1, IgG2, IgG4, and a combination thereof, and whereinthe second CH₃ amino acid sequence comprises a modification that reducesor eliminates binding for the second CH₃ amino acid sequence to ProteinA (see, for example, US 2010/0331527A1, incorporated by reference hereinin its entirety).

In one embodiment, the second CH₃ amino acid sequence comprises an H95Rmodification (by IMGT exon numbering; H435R by EU numbering). In oneembodiment the second CH₃ amino acid sequence further comprises an Y96Fmodification (by IMGT exon numbering; H436F by EU). In anotherembodiment, the second CH₃ amino acid sequence comprises both an H95Rmodification (by IMGT exon numbering; H435R by EU numbering) and an Y96Fmodification (by IMGT exon numbering; H436F by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG1 and further comprises a mutation selected from the groupconsisting of D16E, L18M, N44S, K52N, V57M, and V82I (IMGT; D356E, L38M,N384S, K392N, V397M, and V422I by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG2 and further comprises a mutation selected from the groupconsisting of N44S, K52N, and V82I (IMGT: N384S, K392N, and V422I byEU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG4 and further comprises a mutation selected from the groupconsisting of Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (IMGT: Q355R,N384S, K392N, V397M, R409K, E419Q, and V422I by EU).

In one embodiment, the heavy chain constant region amino acid sequenceis a non-human constant region amino acid sequence, and the heavy chainconstant region amino acid sequence comprises one or more of any of thetypes of modifications described above.

In one embodiment, the heavy chain constant region nucleotide sequenceis a human heavy chain constant region amino acid sequence, and thehuman heavy chain constant region amino acid sequence comprises one ormore of any of the types of modifications described above.

In one embodiment, all or substantially all endogenous V_(H), D, andJ_(H) gene segments are deleted from an immunoglobulin heavy chain locusor rendered non-functional (e.g., via insertion of a nucleotide sequence(e.g., an exogenous nucleotide sequence) in the immunoglobulin locus orvia non-functional rearrangement, or inversion, of the endogenous V_(H),D, J_(H) segments). In one embodiment, e.g., about 80% or more, about85% or more, about 90% or more, about 95% or more, about 96% or more,about 97% or more, about 98% or more, or about 99% or more of allendogenous V_(H), D, or J_(H) gene segments are deleted or renderednon-functional. In one embodiment, e.g., at least 95%, 96%, 97%, 98%, or99% of endogenous functional V, D, or J gene segments are deleted orrendered non-functional.

In one embodiment, the genetically modified immunoglobulin heavy chainlocus comprises a modification that deletes or renders, all orsubstantially all, non-functional endogenous V_(H), D, and J_(H) genesegments; and the genetically modified locus comprises an unrearrangedheavy chain variable region nucleotide sequence comprising at least oneinverted human D gene segment as described herein wherein theunrearranged heavy chain variable region nucleotide sequence is presentat an endogenous location (i.e., where the nucleotide sequence islocated in a wild-type non-human animal) or present ectopically (e.g.,at a locus different from the endogenous immunoglobulin chain locus inits genome, or within its endogenous locus, e.g., within animmunoglobulin variable locus, wherein the endogenous locus is placed ormoved to a different location in the genome).

In one embodiment, the genetically modified immunoglobulin locuscomprises an endogenous Adam6a gene, Adam6b gene, or both, and thegenetic modification does not affect the expression and/or function ofthe endogenous Adam6a gene, Adam6b gene, or both.

In one embodiment, the genetically modified immunoglobulin locuscomprises an ectopically present Adam6a gene, Adam6b gene, or both. Inone embodiment, the Adam6a gene is a non-human Adam6a gene. In oneembodiment, the Adam6a gene is a mouse Adam6a gene. In one embodiment,the Adam6a gene is a human Adam6a gene. In one embodiment, the Adam6bgene is a non-human Adam6b gene. In one embodiment, the Adam6b gene is amouse Adam6b gene. In one embodiment, the Adam6b gene is a human Adam6bgene.

In one embodiment, the genetically modified immunoglobulin locus furthercomprises a humanized, unrearranged λ and/or κ light chain variable genesequence. In one embodiment, the humanized, unrearranged λ and/or κlight chain variable gene sequence is operably linked to animmunoglobulin light chain constant region nucleotide sequence selectedfrom a λ light chain constant region nucleotide sequence and a κ lightchain constant region nucleotide sequence. In one embodiment, thehumanized, unrearranged λ light chain variable region nucleotidesequence is operably linked to a λ light chain constant regionnucleotide sequence. In one embodiment, the λ light chain constantregion nucleotide sequence is a mouse, rat, or human sequence. In oneembodiment, the humanized, unrearranged κ light chain variable regionnucleotide sequence is operably linked to a κ light chain constantregion nucleotide sequence. In one embodiment, the κ light chainconstant region nucleotide sequence is a mouse, rat, or human sequence.

In one embodiment, the genetically modified immunoglobulin locuscomprises an unrearranged light chain variable gene sequence thatcontains at least one modification that introduces at least onehistidine codon in at least one reading frame encoding a light chainvariable domain. In one embodiment, the genetically modifiedimmunoglobulin locus comprises a rearranged (e.g., a rearranged λ or κV/J sequence) sequence that comprises one, two, three, or four codonsfor histidine in a light chain CDR. In one embodiment, the CDR is aselected from a CDR1, CDR2, CDR3, and a combination thereof. In oneembodiment, the unrearranged or rearranged light chain variable regionnucleotide sequence is an unrearranged or rearranged human λ or κ lightchain variable region nucleotide sequence. In one embodiment, theunrearranged or rearranged human λ or κ light chain variable regionnucleotide sequence is present at an endogenous mouse immunoglobulinlight chain locus. In one embodiment, the mouse immunoglobulin lightchain locus is a mouse κ locus. In one embodiment, the mouseimmunoglobulin light chain locus is a mouse immunoglobulin light chainlocus is a mouse λ locus.

In one embodiment, the genetically modified immunoglobulin locus asdescribed herein is present in an immunoglobulin heavy chain locus of amouse. In one embodiment, the genetically modified immunoglobulin locusis present in a humanized immunoglobulin heavy chain locus in aVELOCIMMUNE® mouse.

In one embodiment, the non-human animal is heterozygous for thegenetically modified immunoglobulin heavy chain locus, and the non-humananimal is capable of expressing the human immunoglobulin heavy chainvariable domain comprising at least one histidine residue derivedpredominantly from the genetically modified immunoglobulin heavy chainlocus as described herein.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein exhibits a weaker antigen bindingat an acidic environment (e.g., at a pH of about 5.5 to about 6.0) thana corresponding wild-type heavy chain variable domain without thegenetic modification.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 2 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 25° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin heavy chain locus as described herein has adissociative half-life (t_(1/2)) of less than 2 min at an acidic pH(e.g., pH of about 5.5 to about 6.0) at 37° C. In one embodiment, anantigen-binding protein comprising a heavy chain variable domainexpressed by the genetically modified immunoglobulin heavy chain locusas described herein has a dissociative half-life (t_(1/2)) of less than1 min at an acidic pH (e.g., pH of about 5.5 to about 6.0) at 25° C. Inone embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 1 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 37° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin locus as described herein has at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold, at least about 25-fold, or at least about 30-fold decrease indissociative half-life (t_(1/2)) at an acidic pH (e.g., pH of about 5.5to about 6.0) as compared to the dissociative half-life (t_(1/2)) of theantigen-binding protein at a neutral pH (e.g., pH of about 7.0 to about7.4).

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinlocus as described herein is characterized by improved pH-dependentrecyclability, enhanced serum half-life, or both as compared with awild-type antigen-binding protein without the genetic modification.

In one embodiment, the genetically modified immunoglobulin locusdescribed herein comprises a B cell population that, upon stimulationwith an antigen of interest, is capable of producing antigen-bindingproteins, e.g., antibodies, comprising a heavy chain variable domaincomprising one or more histidine residues. The antigen-binding proteinsas described herein when administered into a subject, exhibits anincreased serum half-life over a corresponding wild-type antigen-bindingprotein, which possesses a similar or sufficiently similar amino acidsequence that encodes the heavy chain variable domain but does notcomprise a histidine residue in the heavy chain variable domain. In someembodiments, the antigen-binding protein described herein exhibits anincreased serum half-life that is at least about 2-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold higher than the corresponding wild-type antigen-binding protein,which possesses a similar or sufficiently similar amino acid sequencethat encodes the heavy chain variable domain but does not comprise ahistidine residue in the heavy chain variable domain.

In one aspect, a non-human animal that is capable of expressing anantigen-binding protein with enhanced pH-dependent recyclability and/orenhanced serum half-life are provided, wherein the non-human animalcomprises in its germline genome an unrearranged human immunoglobulinheavy chain variable region nucleotide sequence, wherein theunrearranged heavy chain variable region nucleotide sequence comprisesan addition of least one histidine codon or a substitution of at leastone endogenous non-histidine codon with a histidine codon as describedherein.

In one embodiment, the antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein exhibits a weaker antigen bindingat an acidic environment (e.g., at a pH of about 5.5 to about 6.0) thana corresponding wild-type heavy chain variable domain without thegenetic modification.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 2 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 25° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin heavy chain locus as described herein has adissociative half-life (t_(1/2)) of less than 2 min at an acidic pH(e.g., pH of about 5.5 to about 6.0) at 37° C. In one embodiment, anantigen-binding protein comprising a heavy chain variable domainexpressed by the genetically modified immunoglobulin heavy chain locusas described herein has a dissociative half-life (t_(1/2)) of less than1 min at an acidic pH (e.g., pH of about 5.5 to about 6.0) at 25° C. Inone embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 1 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 37° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin locus as described herein has at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold, at least about 25-fold, or at least about 30-fold decrease indissociative half-life (t_(1/2)) at an acidic pH (e.g., pH of about 5.5to about 6.0) as compared to the dissociative half-life (t_(1/2)) of theantigen-binding protein at a neutral pH (e.g., pH of about 7.0 to about7.4).

In one embodiment, the antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinlocus as described herein is characterized by improved pH-dependentrecyclability, enhanced serum half-life, or both as compared with awild-type antigen-binding protein without the genetic modification.

In one embodiment, the genetically modified immunoglobulin locusdescribed herein comprises a B cell population that, upon stimulationwith an antigen of interest, is capable of producing antigen-bindingproteins, e.g., antibodies, comprising a heavy chain variable domaincomprising one or more histidine residues. The antigen-binding proteinsas described herein when administered into a subject, exhibits anincreased serum half-life over a corresponding wild-type antigen-bindingprotein, which possesses a similar or sufficiently similar amino acidsequence that encodes the heavy chain variable domain but does notcomprise a histidine residue in the heavy chain variable domain. In someembodiments, the antigen-binding protein described herein exhibits anincreased serum half-life that is at least about 2-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold higher than the corresponding wild-type antigen-binding protein,which possesses a similar or sufficiently similar amino acid sequencethat encodes the heavy chain variable domain but does not comprise ahistidine residue in the heavy chain variable domain.

In one aspect, a targeting construct is provided, comprising 5′ and 3′targeting arms homologous to a genomic D region or genomic V and Jregion of a non-human animal, wherein at least one V_(H), D, or J_(H)gene segment comprises any of the modifications as described herein,e.g., an addition of at least one histidine codon, a substitution of atleast one endogenous non-histidine codon into a histidine codon, and/orinversion of at least one functional D gene segment with respect to acorresponding wild type sequence.

In one aspect, a hybridoma or quadroma is provided that is derived froma cell of any of the non-human animal as described herein. In oneembodiment, the non-human animal is a rodent, e.g., a mouse, a rat, or ahamster.

In one aspect, pluripotent, induced pluripotent, or totipotent stemcells derived form a non-human animal comprising the various genomicmodifications of the described invention are provided. In a specificembodiment, the pluripotent, induced pluripotent, or totipotent stemcells are mouse or rat embryonic stem (ES) cells. In one embodiment, thepluripotent, induced pluripotent, or totipotent stem cells have an XXkaryotype or an XY karyotype. In one embodiment, the pluripotent orinduced pluripotent stem cells are hematopoietic stem cells.

In one aspect, cells that comprise a nucleus containing a geneticmodification as described herein are also provided, e.g., a modificationintroduced into a cell by pronuclear injection. In one embodiment, thepluripotent, induced pluripotent, or totipotent stem cells comprise agenetically modified immunoglobulin genomic locus, wherein the genomiclocus comprises, from 5′ to 3′, (1) an FRT recombination site, (2) humanV_(H) gene segments, (3) a mouse adam6 gene, (4) a loxP recombinationsite, (5) histidine-substituted human D gene segments, (6) human J_(H)gene segments, followed by (7) a mouse E_(i) (intronic enhancer), and(8) a mouse IgM constant region nucleotide sequence.

In one aspect, a lymphocyte isolated from a genetically modifiednon-human animal as described herein is provided. In one embodiment, thelymphocyte is a B cell, wherein the B cell comprises an immunoglobulingenomic locus comprising an unrearranged heavy chain variable regionnucleotide sequence wherein the unrearranged heavy chain variable genesequence comprises an addition of least one histidine codon or asubstitution of at least one endogenous non-histidine codon with ahistidine codon.

In one aspect, a lymphocyte isolated from a genetically modifiednon-human animal as described herein is provided. In one embodiment, thelymphocyte is a B cell, wherein the B cell comprises an immunoglobulinlocus that comprises a human V, D, and J gene segment, wherein at leastone of the human D gene segment has been inverted 5′ to 3′ with respectto wild-type sequences, and wherein at least one reading frame of theinverted human D gene segment encodes at least one histidine residue. Inone embodiment, the B cell is capable of producing an antigen-bindingprotein comprising the genetically modified heavy chain variable domainas described herein. In one embodiment, the genetically modified heavychain variable domain as described herein is operably linked to a heavychain constant region amino acid sequence.

In one aspect, a B cell population is provided that are capable ofexpressing an antigen-binding protein comprising at least one histidineresidue in a heavy chain variable domain, wherein the B cell populationcomprises any genetic modifications as described herein. In oneembodiment, the at least one histidine residue is present in a heavychain CDR. In one embodiment, the CDR is a selected from a CDR1, CDR2,CDR3, and a combination thereof. In one embodiment, the at least onehistidine residue is present in CDR3.

In one aspect, a B cell population is provided that are capable ofexpressing an antigen-binding protein with enhanced serum half-lifeand/or enhanced pH-dependent recyclability, wherein the B cellpopulation comprises any genetic modifications as described herein.

In one aspect, a method for making a non-human animal comprising agenetically modified immunoglobulin heavy chain variable locus isprovided, comprising:

(a) modifying a genome of a non-human animal to delete or rendernon-functional endogenous immunoglobulin heavy chain V, D, and J genesegments (e.g., via insertion of a nucleotide sequence, e.g., anexogenous nucleotide sequence, in the immunoglobulin locus or vianon-functional rearrangement or inversion of endogenous V_(H), D, J_(H)segments); and

(b) placing in the genome an unrearranged heavy chain variable regionnucleotide sequence, wherein the unrearranged heavy chain variableregion nucleotide sequence comprises an addition of least one histidinecodon or a substitution of at least one endogenous non-histidine codonwith a histidine codon as described herein.

In one embodiment, the non-human animal is a mammal, including a rodent,e.g., a mouse, a rat, or a hamster.

In one embodiment, 2 or more, 3 or more, 4 or more, 5 or more, 6 ormore, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 ormore, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 ormore, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 ormore, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 ormore, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 ormore, 37 or more, 38 or more, 39 or more, 40 or more, 41 or more, 42 ormore, 43 or more, 44 or more, 45 or more, 46 or more, 47 or more, 48 ormore, 49 or more, 50 or more, 51 or more, 52 or more, 53 or more, 54 ormore, 55 or more, 56 or more, 57 or more, 58 or more, 59 or more, 60 ormore, or 61 or more of the endogenous non-histidine codons are replacedwith histidine codons.

In one embodiment, the endogenous non-histone codon encodes the aminoacid selected from Y, N, D, Q, S, W, and R.

In one embodiment, the added or substituted histidine codon is presentin an unrearranged heavy chain variable region nucleotide sequence thatencodes an immunoglobulin variable domain selected from an N-terminalregion, a loop 4 region, a CDR1, a CDR2, a CDR3, a combination thereof.

In one embodiment, the added substituted histidine codon histidine codonis present in an unrearranged heavy chain variable region nucleotidesequence that encodes a complementary determining region (CDR) selectedfrom a CDR1, a CDR2, a CDR3, and a combination thereof.

In one embodiment, the added or substituted histidine codon is presentin an unrearranged heavy chain variable region nucleotide sequence thatencodes a frame region (FR) selected from FR1, FR2, FR3, FR4, and acombination thereof.

In one embodiment, the unrearranged heavy chain variable regionnucleotide sequence comprises a genetically modified human V_(H) genesegment, wherein one or more endogenous non-histidine codon in at leastone reading frame of the human V_(H) gene segment has been replaced witha histidine codon.

In one embodiment, the human unrearranged heavy chain variable regionnucleotide sequence comprises a modification that replaces at least oneendogenous non-histidine codon of a human V_(H) gene segment with ahistidine codon, wherein the human V_(H) gene segment is selected fromthe group consisting of V_(H)1-2, V_(H)1-3, V_(H)1-8, V_(H)1-18,V_(H)1-24, V_(H)1-45, V_(H)1-46, V_(H)1-58, V_(H)1-69, V_(H)2-5,V_(H)2-26, V_(H)2-70, V_(H)3-7, V_(H)3-9, V_(H)3-11, V_(H)3-13,V_(H)3-15, V_(H)3-16, V_(H)3-20, V_(H)3-21, V_(H)3-23, V_(H)3-30,V_(H)3-30-3, V_(H) 3-30-5, V_(H)3-33, V_(H)3-35, V_(H)3-38, V_(H)3-43,V_(H)3-48, V_(H)3-49, V_(H)3-53, V_(H)3-64, V_(H)3-66, V_(H)3-72,V_(H)3-73, V_(H)3-74, V_(H)4-4, V_(H)4-28, V_(H)4-30-1, V_(H)4-30-2,V_(H)4-30-4, V_(H)4-31, V_(H)4-34, V_(H)4-39, V_(H)4-59, V_(H)4-61,V_(H)5-51, V_(H)6-1, V_(H)7-4-1, V_(H)7-81, and a combination thereof.

In one embodiment, the human unrearranged heavy chain variable regionnucleotide sequence comprises a genetically modified human J_(H) genesegment, wherein one or more endogenous non-histidine codon in at leastone reading frame of the human J_(H) gene segment has been replaced witha histidine codon.

In one embodiment, the human unrearranged heavy chain variable regionnucleotide sequence comprises a modification that replaces at least oneendogenous non-histidine codon of a human J_(H) segment with a histidinecodon, wherein the human J_(H) gene segment is selected from the groupconsisting of J_(H)1, J_(H)2, 43, 44, 45, J_(H)6, and a combinationthereof.

In one embodiment, the added or substituted histidine codon is presentin a heavy chain variable region nucleotide sequence that encodes partof a CDR3. In one embodiment, the part of CDR3 comprises an amino acidsequence derived from a reading frame of a genetically modified human Dgene segment comprising a modification that replaces at least oneendogenous non-histidine codon in the reading frame with a histidinecodon.

In one embodiment, the endogenous non-histidine codon that issubstituted with a histidine codon encodes the amino acid selected fromY, N, D, Q, S, W, and R.

In one embodiment, the added or substituted histidine codon is presentin at least one reading frame of the human D gene segment that is mostfrequently observed in VELOCIMMUNE® humanized immunoglobulin mice.

In one embodiment, the reading frame of the genetically modified human Dgene segment that encodes part of CDR3 is selected from a hydrophobicframe, a stop frame, and a hydrophilic frame.

In one embodiment, the reading frame is a hydrophobic frame of a human Dgene segment.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D1-1 (GTTGT; SEQ ID NO: 88), D1-7(GITGT; SEQ ID NO: 89), D1-20 (GITGT; SEQ ID NO: 89), and D1-26 (GIVGAT;SEQ ID NO:90), and the human D gene segment further comprises amodification that replaces at least one endogenous non-histidine codonin the nucleotide sequence with a histidine codon.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D2-2 (DIVVVPAAI; SEQ ID NO:92),D2-8 (DIVLMVYAI; SEQ ID NO: 94), D2-15 (DIVVVVAAT; SEQ ID NO:95), andD2-21 (HIVVVTAI; SEQ ID NO: 97), and the human D gene segment furthercomprises a modification that replaces at least one endogenousnon-histidine codon in the nucleotide sequence with a histidine codon.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D3-3 (ITIFGVVII; SEQ ID NO:98),D3-9 (ITIF*LVII; SEQ ID NO:99, SEQ ID NO:100), D3-10 (ITMVRGVII; SEQ IDNO:101), D3-16 (IMITFGGVIVI; SEQ ID NO:102), and D3-22 (ITMIVVVIT; SEQID NO:103), and the human D gene segment further comprises amodification that replaces at least one endogenous codon in thenucleotide sequence with a histidine codon.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D4-4 (TTVT; SEQ ID NO:105), D4-11(TTVT; SEQ ID NO:105), D4-17 (TTVT; SEQ ID NO:105), D4-23 (TTVVT; SEQ IDNO: 106) and the human D gene segment further comprises a modificationthat replaces at least one endogenous non-histidine codon in thenucleotide sequence with a histidine codon.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D5-5 (VDTAMV; SEQ ID NO: 107),D5-12 (VDIVATI; SEQ ID NO:108), D5-18 (VDTAMV; SEQ ID NO:107), and D5-24(VEMATI; SEQ ID NO:109), and the human D gene segment further comprisesa modification that replaces at least one endogenous non-histidine codonin the nucleotide sequence with a histidine codon.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D6-6 (SIAAR; SEQ ID NO:111), D6-13(GIAAAG; SEQ ID NO:113), and D6-19 (GIAVAG; SEQ ID NO:115), and thehuman D gene segment further comprises a modification that replaces atleast one endogenous non-histidine codon in the nucleotide sequence witha histidine codon.

In one embodiment, the hydrophobic frame comprises a nucleotide sequencethat encodes human D7-27 (LTG), and the human D gene segment furthercomprises a modification that replaces at least one endogenousnon-histidine codon in the nucleotide sequence with a histidine codon.

In one embodiment, the reading frame is a stop reading frame of a humanD gene segment.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D1-1 (VQLER; SEQ ID NO:8),D1-7(V*LEL), D1-20(V*LER), D1-26 (V*WELL; SEQ ID NO:12), and the human Dgene segment further comprises a modification that replaces at least oneendogenous non-histidine codon in the nucleotide sequence with ahistidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D2-2 (RIL**YQLLY; SEQ ID NO:14),D2-8 (RILY*WCMLY; SEQ ID NO:16 and SEQ ID NO: 17), D2-15 (RIL*WW*LLL),and D2-21 (SILWW*LLF; SEQ ID NO:19), and the human D gene segmentfurther comprises a modification that replaces at least one endogenousnon-histidine codon in the nucleotide sequence with a histidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D3-3 (VLRFLEWLLY; SEQ ID NO:21),D3-9 (VLRYFDWLL*; SEQ ID NO:23), D3-10 (VLLWFGELL*; SEQ ID NO:25), D3-16(VL*LRLGELSLY; SEQ ID NO:27), and D3-22 (VLL***WLLL; SEQ ID NO:29), andthe human D gene segment comprises a modification that replaces at leastone endogenous non-histidine codon in the nucleotide sequence with ahistidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D4-4 (*LQ*L), D4-11 (*LQ*L), D4-17(*LR*L), and D4-23 (*LRW*L), and the human D gene segment comprises amodification that replaces at least one endogenous non-histidine codonin the nucleotide sequence with a histidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D5-5 (WIQLWL; SEQ ID NO:35); D5-12(Wl*WLRL; SEQ ID NO:37), D5-18 (WIQLWL; SEQ ID NO:35), and D5-24(*RWLQL; SEQ ID NO:39), and the human D gene segment comprises amodification that replaces at least one endogenous non-histidine codonin the nucleotide sequence with a histidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D6-6 (V*QLV), D6-13 (V*QQLV; SEQID NO:41), and D6-19 (V*QWLV; SEQ ID NO:43), and the human D genesegment further comprises a modification that replaces at least oneendogenous non-histidine codon in the nucleotide sequence with ahistidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes D7-27 (*LG), and the humanD gene segment further comprises a modification that replaces at leastone endogenous codon of the human D gene segment in the nucleotidesequence with a histidine codon.

In one embodiment, the reading frame is a hydrophilic frame of a human Dgene segment.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D1-1 (YNWND; SEQ ID NO: 45), D1-7(YNWNY; SEQ ID NO: 47), D1-20 (YNWND; SEQ ID NO: 45), and D1-26 (YSGSYY;SEQ ID NO:49), and the human D gene segment further comprises amodification that replaces at least one endogenous codon in thenucleotide sequence with a histidine codon. In one embodiment, thehydrophilic frame comprises a nucleotide sequence that encodes the aminoacid sequence selected from the group consisting of SEQ ID NO: 46, SEQID NO: 48, SEQ ID NO: 50, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D2-2 (GYCSSTSCYT; SEQ ID NO:51),D2-8 (GYCTNGVCYT; SEQ ID NO: 53), D2-15 (GYCSGGSCYS; SEQ ID NO:55), andD2-21 (AYCGGDCYS; SEQ ID NO:57), and the human D gene segment furthercomprises a modification that replaces at least one endogenous codon inthe nucleotide sequence with a histidine codon. In one embodiment, thehydrophilic frame comprises a nucleotide sequence that encodes the aminoacid sequence selected from the group consisting of SEQ ID NO: 52, SEQID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D3-3 (YYDFWSGYYT; SEQ ID NO:59),D3-9 (YYDILTGYYN; SEQ ID NO:61), D3-10 (YYYGSGSYYN; SEQ ID NO:63), D3-16(YYDYVWGSYRYT; SEQ ID NO:65), and D3-22 (YYYDSSGYYY; SEQ ID NO:67), andthe human D gene segment further comprises a modification that replacesat least one endogenous codon in the nucleotide sequence with ahistidine codon. In one embodiment, the hydrophilic frame comprises anucleotide sequence encodes the amino acid sequence selected from thegroup consisting of SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ IDNO: 66, SEQ ID NO: 68, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D4-4 (DYSNY; SEQ ID NO:69), D4-11(DYSNY; SEQ ID NO:69), D4-17 (DYGDY; SEQ ID NO:71), and D4-23 (DYGGNS;SEQ ID NO:73), and the human D gene segment comprises a modificationthat replaces at least one endogenous codon in the nucleotide sequencewith a histidine codon. In one embodiment, the hydrophilic framecomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of SEQ ID NO: 70, SEQ ID NO: 72, SEQID NO: 74, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D5-5 (GYSYGY; SEQ ID NO:75), D5-12(GYSGYDY; SEQ ID NO:77), D5-18 (GYSYGY; SEQ ID NO:75), and D5-24(RDGYNY; SEQ ID NO:79), and the human D gene segment further comprises amodification that replaces at least one endogenous codon in thenucleotide sequence with a histidine codon. In one embodiment, thehydrophilic frame comprises a nucleotide sequence that encodes the aminoacid sequence selected from the group consisting of SEQ ID NO: 76, SEQID NO: 78, SEQ ID NO: 80, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D6-6 (EYSSSS; SEQ ID NO: 81),D6-13 (GYSSSWY; SEQ ID NO:83), and D6-19 (GYSSGWY; SEQ ID NO:85), andthe human D gene segment further comprises a modification that replacesat least one endogenous codon in the nucleotide sequence with ahistidine codon. In one embodiment, the hydrophilic frame comprises anucleotide sequence that encodes the amino acid sequence selected fromthe group consisting of SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQID NO: 76, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes D7-27 (NWG), and the humanD gene segment further comprises a modification that replaces at leastone endogenous codon in the nucleotide sequence a histidine codon.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 48, SEQID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO:68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ IDNO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, anda combination thereof.

In one embodiment, the unrearranged heavy chain variable regionnucleotide sequence comprising the inverted human D gene segment isoperably linked to a human or non-human heavy chain constant regionnucleotide sequence that encodes an immunoglobulin isotype selected fromIgM, IgD, IgG, IgE, and IgA.

In one embodiment, the human unrearranged immunoglobulin heavy chainvariable region nucleotide sequence is operably linked to a human ornon-human heavy chain constant region nucleotide sequence selected froma C_(H)1, a hinge, a C_(H)2, a C_(H)3, and a combination thereof. In oneembodiment, the heavy chain constant region nucleotide sequencecomprises a C_(H)1, a hinge, a C_(H)2, and a C_(H)3 (i.e.,C_(H)1-hinge-C_(H)2-C_(H)3).

In one embodiment, a heavy chain constant region nucleotide sequence ispresent at an endogenous locus (i.e., where the nucleotide sequence islocated in a wild-type non-human animal) or present ectopically (e.g.,at a locus different from the endogenous immunoglobulin chain locus inits genome, or within its endogenous locus, e.g., within animmunoglobulin variable locus, wherein the endogenous locus is placed ormoved to a different location in the genome).

In one embodiment, the heavy chain constant region nucleotide sequencecomprises a modification in a C_(H)2 or a C_(H)3, wherein themodification increases the affinity of the heavy chain constant regionamino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a modification at position 250 (e.g., E or Q); 250 and 428(e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256(e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433(e.g., L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification atposition 250 and/or 428; or a modification at position 307 or 308 (e.g.,308F, V308F), and 434. In one embodiment, the modification comprises a428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I(e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K)and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y,254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Qand M428L); and a 307 and/or 308 modification (e.g., 308F or 308P),wherein the modification increases the affinity of the heavy chainconstant region amino acid sequence to FcRn in an acidic environment(e.g., in an endosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 252 and 257,wherein the modification increases the affinity of the human C_(H)2amino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 307 and 311,wherein the modification increases the affinity of the C_(H)2 amino acidsequence to FcRn in an acidic environment (e.g., in an endosome where pHranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)3 amino acid sequence, wherein the C_(H)3 aminoacid sequence comprises at least one modification between amino acidresidues at positions 433 and 436, wherein the modification increasesthe affinity of the C_(H)3 amino acid sequence to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,N434S, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,V259I, V308F, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising an N434A mutation.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M252Y,S254T, T256E, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of T250Q,M248L, or both.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of H433K,N434Y, or both.

In one embodiment, the genetically modified immunoglobulin locuscomprises: (1) a first allele, wherein the unrearranged humanimmunoglobulin heavy chain variable region nucleotide sequence asdescribed herein is operably linked to a first heavy chain constantregion nucleotide sequence encoding a first CH₃ amino acid sequence of ahuman IgG selected from IgG1, IgG2, IgG4, and a combination thereof; and(2) a second allele, wherein the unrearranged human immunoglobulin heavychain variable region nucleotide sequence as described herein isoperably linked to a second heavy chain constant region nucleotidesequence encoding a second C_(H)3 amino acid sequence of the human IgGselected from IgG1, IgG2, IgG4, and a combination thereof, and whereinthe second CH₃ amino acid sequence comprises a modification that reducesor eliminates binding for the second CH₃ amino acid sequence to ProteinA (see, for example, US 2010/0331527A1, which is incorporated byreference herein in its entirety).

In one embodiment, the second CH₃ amino acid sequence comprises an H95Rmodification (by IMGT exon numbering; H435R by EU numbering). In oneembodiment the second CH₃ amino acid sequence further comprises an Y96Fmodification (by IMGT exon numbering; H436F by EU). In anotherembodiment, the second CH₃ amino acid sequence comprises both an H95Rmodification (by IMGT exon numbering; H435R by EU numbering) and an Y96Fmodification (by IMGT exon numbering; H436F by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG1 and further comprises a mutation selected from the groupconsisting of D16E, L18M, N44S, K52N, V57M, and V82I (IMGT; D356E, L38M,N384S, K392N, V397M, and V422I by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG2 and further comprises a mutation selected from the groupconsisting of N44S, K52N, and V82I (IMGT: N384S, K392N, and V422I byEU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG4 and further comprises a mutation selected from the groupconsisting of Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (IMGT: Q355R,N384S, K392N, V397M, R409K, E419Q, and V422I by EU).

In one embodiment, the heavy chain constant region amino acid sequenceis a non-human constant region amino acid sequence, and the heavy chainconstant region amino acid sequence comprises one or more of any of thetypes of modifications described above.

In one embodiment, the heavy chain constant region nucleotide sequenceis a human heavy chain constant region amino acid sequence, and thehuman heavy chain constant region amino acid sequence comprises one ormore of any of the types of modifications described above.

In one embodiment, all or substantially all endogenous V_(H), D, andJ_(H) gene segments are deleted from an immunoglobulin heavy chain locusor rendered non-functional (e.g., via insertion of a nucleotide sequence(e.g., an exogenous nucleotide sequence) in the immunoglobulin locus orvia non-functional rearrangement, or inversion, of the endogenous V_(H),D, J_(H) segments). In one embodiment, e.g., about 80% or more, about85% or more, about 90% or more, about 95% or more, about 96% or more,about 97% or more, about 98% or more, or about 99% or more of allendogenous V_(H), D, or J_(H) gene segments are deleted or renderednon-functional. In one embodiment, e.g., at least 95%, 96%, 97%, 98%, or99% of endogenous functional V, D, or J gene segments are deleted orrendered non-functional.

In one embodiment, the genetically modified locus comprises amodification that deletes or renders non-functional all or substantiallyall endogenous V_(H), D, and J_(H) gene segments; and the genomic locuscomprises the genetically modified, unrearranged human heavy chainvariable region nucleotide sequence comprising a substitution of atleast one endogenous non-histidine codon with a histidine codon in atleast one reading frame. In one embodiment, the genetically modified,unrearranged immunoglobulin heavy chain variable gene sequence ispresent at an endogenous location (i.e., where the nucleotide sequenceis located in a wild-type non-human animal) or present ectopically(e.g., at a locus different from the endogenous immunoglobulin chainlocus in its genome), or within its endogenous locus, e.g., within animmunoglobulin variable locus, wherein the endogenous locus is placed ormoved to a different location in the genome.

In one embodiment, the genetically modified locus comprises anendogenous Adam6a gene, Adam6b gene, or both, and the geneticmodification does not affect the expression and/or function of theendogenous Adam6a gene, Adam6b gene, or both.

In one embodiment, the genetically modified locus comprises anectopically present Adam6a gene, Adam6b gene, or both. In oneembodiment, the Adam6a gene is a non-human Adam6a gene. In oneembodiment, the Adam6a gene is a mouse Adam6a gene. In one embodiment,the Adam6a gene is a human Adam6a gene. In one embodiment, the Adam6bgene is a non-human Adam6b gene. In one embodiment, the Adam6b gene is amouse Adam6b gene. In one embodiment, the Adam6b gene is a human Adam6bgene.

In one embodiment, the genetically modified immunoglobulin locus furthercomprises a humanized, unrearranged λ and/or κ light chain variable genesequence. In one embodiment, the humanized, unrearranged λ and/or κlight chain variable gene sequence is operably linked to animmunoglobulin light chain constant region nucleotide sequence selectedfrom a λ light chain constant region nucleotide sequence and a κ lightchain constant region nucleotide sequence. In one embodiment, thehumanized, unrearranged λ light chain variable region nucleotidesequence is operably linked to a λ light chain constant regionnucleotide sequence. In one embodiment, the λ light chain constantregion nucleotide sequence is a mouse, rat, or human sequence. In oneembodiment, the humanized, unrearranged κ light chain variable regionnucleotide sequence is operably linked to a κ light chain constantregion nucleotide sequence. In one embodiment, the κ light chainconstant region nucleotide sequence is a mouse, rat, or human sequence.

In one embodiment, the genetically modified immunoglobulin locuscomprises an unrearranged light chain variable gene sequence thatcontains at least one modification that introduces at least onehistidine codon in at least one reading frame encoding a light chainvariable domain. In one embodiment, the genetically modifiedimmunoglobulin locus comprises a rearranged (e.g., a rearranged λ or κV/J sequence) sequence that comprises one, two, three, or four codonsfor histidine in a light chain CDR. In one embodiment, the CDR is aselected from a CDR1, CDR2, CDR3, and a combination thereof. In oneembodiment, the unrearranged or rearranged light chain variable regionnucleotide sequence is an unrearranged or rearranged human λ or κ lightchain variable region nucleotide sequence. In one embodiment, theunrearranged or rearranged human λ or κ light chain variable regionnucleotide sequence is present at an endogenous mouse immunoglobulinlight chain locus. In one embodiment, the mouse immunoglobulin lightchain locus is a mouse κ locus. In one embodiment the mouseimmunoglobulin light chain locus is a mouse λ locus.

In one embodiment, the genetically modified immunoglobulin locus asdescribed herein is present in an immunoglobulin heavy chain locus of amouse. In one embodiment, the genetically modified immunoglobulin locusis present in a humanized immunoglobulin heavy chain locus in aVELOCIMMUNE® mouse.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein exhibits a weaker antigen bindingat an acidic environment (e.g., at a pH of about 5.5 to about 6.0) thana corresponding wild-type heavy chain variable domain without thegenetic modification.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 2 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 25° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin heavy chain locus as described herein has adissociative half-life (t_(1/2)) of less than 2 min at an acidic pH(e.g., pH of about 5.5 to about 6.0) at 37° C. In one embodiment, anantigen-binding protein comprising a heavy chain variable domainexpressed by the genetically modified immunoglobulin heavy chain locusas described herein has a dissociative half-life (t_(1/2)) of less than1 min at an acidic pH (e.g., pH of about 5.5 to about 6.0) at 25° C. Inone embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 1 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 37° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin locus as described herein has at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold, at least about 25-fold, or at least about 30-fold decrease indissociative half-life (t_(1/2)) at an acidic pH (e.g., pH of about 5.5to about 6.0) as compared to the dissociative half-life (t_(1/2)) of theantigen-binding protein at a neutral pH (e.g., pH of about 7.0 to about7.4).

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinlocus as described herein is characterized by improved pH-dependentrecyclability, enhanced serum half-life, or both as compared with awild-type antigen-binding protein without the genetic modification.

In one embodiment, the genetically modified immunoglobulin locusdescribed herein comprises a B cell population that, upon stimulationwith an antigen of interest, is capable of producing antigen-bindingproteins, e.g., antibodies, comprising a heavy chain variable domaincomprising one or more histidine residues. The antigen-binding proteinsas described herein when administered into a subject, exhibits anincreased serum half-life over a corresponding wild-type antigen-bindingprotein, which possesses a similar or sufficiently similar amino acidsequence that encodes the heavy chain variable domain but does notcomprise a histidine residue in the heavy chain variable domain. In someembodiments, the antigen-binding protein described herein exhibits anincreased serum half-life that is at least about 2-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold higher than the corresponding wild-type antigen-binding protein,which possesses a similar or sufficiently similar amino acid sequencethat encodes the heavy chain variable domain but does not comprise ahistidine residue in the heavy chain variable domain.

In one aspect, a method for making a non-human animal comprising agenetically modified immunoglobulin heavy chain variable locus isprovided, comprising:

(a) modifying a genome of a non-human animal to delete or rendernon-functional endogenous immunoglobulin heavy chain V, D, and J genesegments (e.g., via insertion of a nucleotide sequence (e.g., anexogenous nucleotide sequence) in the immunoglobulin locus or vianon-functional rearrangement or inversion of endogenous V_(H), D, J_(H)segments); and

(b) placing in the genome a human V_(H), D, and J_(H) gene segment,wherein at least one of the human D gene segment has been inverted 5′ to3′ with respect to a corresponding wild-type sequence, and wherein atleast one reading frame of the inverted human D gene segment comprises ahistidine codon.

In one embodiment, the non-human animal is a mammal, including a rodent,e.g., a mouse, a rat, or a hamster

In one embodiment, the genetically modified immunoglobulin locus ispresent in a germline genome.

In one embodiment, the genetically modified immunoglobulin locus encodesan immunoglobulin heavy chain variable domain comprising one or more, 2or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 ormore, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 ormore, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 ormore, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 ormore, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 ormore, 33 or more, or 34 or more of histidine residues.

In one embodiment, at least two, at least three, at least four, at leastfive, at least six, at least seven, at least eight, at least nine, atleast ten, at least eleven, at least twelve, at least thirteen, at leastfourteen, at least fifteen, at least sixteen, at least seventeen, atleast eighteen, at least nineteen, at least twenty, at least twenty one,at least twenty two, at least twenty three, at least twenty four, or allor substantially all of functional human D gene segments have invertedorientation with respect to corresponding wild type sequences.

In one embodiment, all or substantially all of endogenous immunoglobulinV_(H), D, J_(H) gene segments are deleted from the immunoglobulin heavychain locus or rendered non-functional (e.g., via insertion of anucleotide sequence, e.g., exogenous nucleotide sequence, in theimmunoglobulin locus or via non-functional rearrangement or inversion ofall, or substantially all, endogenous immunoglobulin V_(H), D, J_(H)segments), and the genetically modified immunoglobulin locus comprises ahuman V_(H), D, and J_(H) gene segments, wherein at least one of thehuman D gene segment is present in an inverted orientation with respectto a corresponding wild type sequence, and wherein at least one readingframe in the inverted human D gene segment comprises at least onehistidine codon.

In one embodiment, the inverted human D gene segment is operably linkedto a human V_(H) gene segment, and/or human J_(H) gene segment

In one embodiment, the human D gene segment that is present in theinverted orientation relative to wild type sequences is selected fromthe group consisting of D1-1, D1-7, D1-20, D1-26, D2-2, D2-8, D2-15,D2-21, D3-3, D3-9, D3-10, D3-16, D3-22, D4-4, D4-11, D4-17, D4-23, D5-5,D5-12, D5-18, D5-24, D6-6, D6-13, D6-19, D7-27, and a combinationthereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD1 gene segment selected from the group consisting of D1-1, D1-7, D1-20,D1-26, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD2 gene segment selected from the group consisting of D2-2, D2-8, D2-15,D2-21, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD3 gene segment selected from the group consisting of D3-3, D3-9, D3-10,D3-16, D3-22, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD4 gene segment selected from the group consisting of D4-4, D4-11,D4-17, D4-23, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD5 gene segment selected from the group consisting of D5-5, D5-12,D5-18, D5-24, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD6 gene segment selected from the group consisting of D6-6, D6-13,D6-19, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence isD7-27.

In one embodiment, the reading frame of the human D gene segment isselected from a stop reading frame, a hydrophilic reading frame, ahydrophobic reading frame, and a combination thereof.

In one embodiment, the unrearranged heavy chain variable regionnucleotide sequence comprising the inverted human D gene segment isoperably linked to a human or non-human heavy chain constant regionnucleotide sequence that encodes an immunoglobulin isotype selected fromIgM, IgD, IgG, IgE, and IgA.

In one embodiment, the human unrearranged immunoglobulin heavy chainvariable region nucleotide sequence is operably linked to a human ornon-human heavy chain constant region nucleotide sequence selected froma C_(H)1, a hinge, a C_(H)2, a C_(H)3, and a combination thereof. In oneembodiment, the heavy chain constant region nucleotide sequencecomprises a C_(H)1, a hinge, a C_(H)2, and a C_(H)3 (i.e.,C_(H)1-hinge-C_(H)2-CH3).

In one embodiment, a heavy chain constant region nucleotide sequence ispresent at an endogenous locus (i.e., where the nucleotide sequence islocated in a wild-type non-human animal) or present ectopically (e.g.,at a locus different from the endogenous immunoglobulin chain locus inits genome, or within its endogenous locus, e.g., within animmunoglobulin variable locus, wherein the endogenous locus is placed ormoved to a different location in the genome).

In one embodiment, the heavy chain constant region nucleotide sequencecomprises a modification in a C_(H)2 or a C_(H)3, wherein themodification increases the affinity of the heavy chain constant regionamino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a modification at position 250 (e.g., E or Q); 250 and 428(e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256(e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433(e.g., L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification atposition 250 and/or 428; or a modification at position 307 or 308 (e.g.,308F, V308F), and 434. In one embodiment, the modification comprises a428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I(e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K)and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y,254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Qand M428L); and a 307 and/or 308 modification (e.g., 308F or 308P),wherein the modification increases the affinity of the heavy chainconstant region amino acid sequence to FcRn in an acidic environment(e.g., in an endosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 252 and 257,wherein the modification increases the affinity of the human C_(H)2amino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 307 and 311,wherein the modification increases the affinity of the C_(H)2 amino acidsequence to FcRn in an acidic environment (e.g., in an endosome where pHranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)3 amino acid sequence, wherein the C_(H)3 aminoacid sequence comprises at least one modification between amino acidresidues at positions 433 and 436, wherein the modification increasesthe affinity of the C_(H)3 amino acid sequence to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,N434S, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,V259I, V308F, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising an N434A mutation.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M252Y,S254T, T256E, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of T250Q,M248L, or both.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of H433K,N434Y, or both.

In one embodiment, the genetically modified immunoglobulin locuscomprises: (1) a first allele, wherein the unrearranged humanimmunoglobulin heavy chain variable region nucleotide sequence asdescribed herein is operably linked to a first heavy chain constantregion nucleotide sequence encoding a first CH₃ amino acid sequence of ahuman IgG selected from IgG1, IgG2, IgG4, and a combination thereof; and(2) a second allele, wherein the unrearranged human immunoglobulin heavychain variable region nucleotide sequence as described herein isoperably linked to a second heavy chain constant region nucleotidesequence encoding a second C_(H)3 amino acid sequence of the human IgGselected from IgG1, IgG2, IgG4, and a combination thereof, and whereinthe second CH₃ amino acid sequence comprises a modification that reducesor eliminates binding for the second CH₃ amino acid sequence to ProteinA (see, for example, US 2010/0331527A1, which is incorporated byreference herein in its entirety).

In one embodiment, the second CH₃ amino acid sequence comprises an H95Rmodification (by IMGT exon numbering; H435R by EU numbering). In oneembodiment the second CH₃ amino acid sequence further comprises an Y96Fmodification (by IMGT exon numbering; H436F by EU). In anotherembodiment, the second CH₃ amino acid sequence comprises both an H95Rmodification (by IMGT exon numbering; H435R by EU numbering) and an Y96Fmodification (by IMGT exon numbering; H436F by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG1 and further comprises a mutation selected from the groupconsisting of D16E, L18M, N44S, K52N, V57M, and V82I (IMGT; D356E, L38M,N384S, K392N, V397M, and V422I by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG2 and further comprises a mutation selected from the groupconsisting of N44S, K52N, and V82I (IMGT: N384S, K392N, and V422I byEU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG4 and further comprises a mutation selected from the groupconsisting of Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (IMGT: Q355R,N384S, K392N, V397M, R409K, E419Q, and V422I by EU).

In one embodiment, the heavy chain constant region amino acid sequenceis a non-human constant region amino acid sequence, and the heavy chainconstant region amino acid sequence comprises one or more of any of thetypes of modifications described above.

In one embodiment, the heavy chain constant region nucleotide sequenceis a human heavy chain constant region amino acid sequence, and thehuman heavy chain constant region amino acid sequence comprises one ormore of any of the types of modifications described above.

In one embodiment, all or substantially all endogenous V_(H), D, andJ_(H) gene segments are deleted from an immunoglobulin heavy chain locusor rendered non-functional (e.g., via insertion of a nucleotide sequence(e.g., an exogenous nucleotide sequence) in the immunoglobulin locus orvia non-functional rearrangement, or inversion, of the endogenous V_(H),D, J_(H) segments). In one embodiment, e.g., about 80% or more, about85% or more, about 90% or more, about 95% or more, about 96% or more,about 97% or more, about 98% or more, or about 99% or more of allendogenous V_(H), D, or J_(H) gene segments are deleted or renderednon-functional. In one embodiment, e.g., at least 95%, 96%, 97%, 98%, or99% of endogenous functional V, D, or J gene segments are deleted orrendered non-functional.

In one embodiment, the genetically modified immunoglobulin heavy chainlocus comprises a modification that deletes or renders, all orsubstantially all, non-functional endogenous V_(H), D, and J_(H) genesegments; and the genetically modified locus comprises an unrearrangedheavy chain variable region nucleotide sequence comprising at least oneinverted human D gene segment as described herein wherein theunrearranged heavy chain variable region nucleotide sequence is presentat an endogenous location (i.e., where the nucleotide sequence islocated in a wild-type non-human animal) or present ectopically (e.g.,at a locus different from the endogenous immunoglobulin chain locus inits genome, or within its endogenous locus, e.g., within animmunoglobulin variable locus, wherein the endogenous locus is placed ormoved to a different location in the genome).

In one embodiment, the genetically modified immunoglobulin locuscomprises an endogenous Adam6a gene, Adam6b gene, or both, and thegenetic modification does not affect the expression and/or function ofthe endogenous Adam6a gene, Adam6b gene, or both.

In one embodiment, the genetically modified immunoglobulin locuscomprises an ectopically present Adam6a gene, Adam6b gene, or both. Inone embodiment, the Adam6a gene is a non-human Adam6a gene. In oneembodiment, the Adam6a gene is a mouse Adam6a gene. In one embodiment,the Adam6a gene is a human Adam6a gene. In one embodiment, the Adam6bgene is a non-human Adam6b gene. In one embodiment, the Adam6b gene is amouse Adam6b gene. In one embodiment, the Adam6b gene is a human Adam6bgene.

In one embodiment, the genetically modified immunoglobulin locus furthercomprises a humanized, unrearranged λ and/or κ light chain variable genesequence. In one embodiment, the humanized, unrearranged λ and/or κlight chain variable gene sequence is operably linked to animmunoglobulin light chain constant region nucleotide sequence selectedfrom a λ light chain constant region nucleotide sequence and a κ lightchain constant region nucleotide sequence. In one embodiment, thehumanized, unrearranged λ light chain variable region nucleotidesequence is operably linked to a λ light chain constant regionnucleotide sequence. In one embodiment, the λ light chain constantregion nucleotide sequence is a mouse, rat, or human sequence. In oneembodiment, the humanized, unrearranged κ light chain variable regionnucleotide sequence is operably linked to a κ light chain constantregion nucleotide sequence. In one embodiment, the κ light chainconstant region nucleotide sequence is a mouse, rat, or human sequence.

In one embodiment, the genetically modified immunoglobulin locuscomprises an unrearranged light chain variable gene sequence thatcontains at least one modification that introduces at least onehistidine codon in at least one reading frame encoding a light chainvariable domain. In one embodiment, the genetically modifiedimmunoglobulin locus comprises a rearranged (e.g., a rearranged λ or κV/J sequence) sequence that comprises one, two, three, or four codonsfor histidine in a light chain CDR. In one embodiment, the CDR is aselected from a CDR1, CDR2, CDR3, and a combination thereof. In oneembodiment, the unrearranged or rearranged light chain variable regionnucleotide sequence is an unrearranged or rearranged human λ or κ lightchain variable region nucleotide sequence. In one embodiment, theunrearranged or rearranged human λ or κ light chain variable regionnucleotide sequence is present at an endogenous mouse immunoglobulinlight chain locus. In one embodiment, the mouse immunoglobulin lightchain locus is a mouse κ locus. In one embodiment, the mouseimmunoglobulin light chain locus is a mouse immunoglobulin light chainlocus is a mouse λ locus.

In one embodiment, the genetically modified immunoglobulin locus asdescribed herein is present in an immunoglobulin heavy chain locus of amouse. In one embodiment, the genetically modified immunoglobulin locusis present in a humanized immunoglobulin heavy chain locus in aVELOCIMMUNE® mouse.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein exhibits a weaker antigen bindingat an acidic environment (e.g., at a pH of about 5.5 to about 6.0) thana corresponding wild-type heavy chain variable domain without thegenetic modification.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 2 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 25° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin heavy chain locus as described herein has adissociative half-life (t_(1/2)) of less than 2 min at an acidic pH(e.g., pH of about 5.5 to about 6.0) at 37° C. In one embodiment, anantigen-binding protein comprising a heavy chain variable domainexpressed by the genetically modified immunoglobulin heavy chain locusas described herein has a dissociative half-life (t_(1/2)) of less than1 min at an acidic pH (e.g., pH of about 5.5 to about 6.0) at 25° C. Inone embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 1 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 37° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin locus as described herein has at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold, at least about 25-fold, or at least about 30-fold decrease indissociative half-life (t_(1/2)) at an acidic pH (e.g., pH of about 5.5to about 6.0) as compared to the dissociative half-life (t_(1/2)) of theantigen-binding protein at a neutral pH (e.g., pH of about 7.0 to about7.4).

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinlocus as described herein is characterized by improved pH-dependentrecyclability, enhanced serum half-life, or both as compared with awild-type antigen-binding protein without the genetic modification.

In one embodiment, the genetically modified immunoglobulin locusdescribed herein comprises an enriched B cell population that, uponstimulation with an antigen of interest, is capable of producingantigen-binding proteins, e.g., antibodies, comprising a heavy chainvariable domain comprising one or more histidine residues. Theantigen-binding proteins as described herein, when administered into asubject, exhibits an increased serum half-life over a correspondingwild-type antigen-binding protein, which possesses a similar orsufficiently similar amino acid sequence that encodes the heavy chainvariable domain but does not comprise a histidine residue in the heavychain variable domain. In some embodiments, the antigen-binding proteindescribed herein exhibits an increased serum half-life that is at leastabout 2-fold, at least about 5-fold, at least about 10-fold, at leastabout 15-fold, at least about 20-fold higher than the correspondingwild-type antigen-binding protein, which possesses a similar orsufficiently similar amino acid sequence that encodes the heavy chainvariable domain but does not comprise a histidine residue in the heavychain variable domain.

In one aspect, a method for making a non-human animal that is capable ofproducing an immunoglobulin heavy chain variable domain with enhancedserum half-life and/or enhanced pH-dependent recyclability is provided,comprising

(a) modifying a genome of a non-human animal to delete or rendernon-functional endogenous immunoglobulin heavy chain V, D, and J genesegments (e.g., via insertion of a nucleotide sequence (e.g., anexogenous nucleotide sequence) in the immunoglobulin locus or vianon-functional rearrangement or inversion of endogenous V_(H), D, J_(H)segments); and

(b) placing in the genome an unrearranged human heavy chain variableregion nucleotide sequence, wherein the unrearranged heavy chainvariable region nucleotide sequence comprises an addition of least onehistidine codon or a substitution of at least one endogenousnon-histidine codon with a histidine codon, and wherein anantigen-binding protein comprising the immunoglobulin heavy chainvariable domain produced by the non-human animal exhibits enhanced serumhalf-life and/or enhanced pH-dependent recyclability as compared to awild-type immunoglobulin heavy chain domain.

In one embodiment, the non-human animal, upon contact with an antigen,can produce an enriched population of B cell repertoire that expressesan antigen-binding protein with enhanced serum half-life and/or enhancedpH-dependent recyclability, wherein the enriched B cell populationcomprises any genetic modifications as described herein.

In one embodiment, an antigen-binding protein produced by thegenetically modified non-human animal is characterized by sufficientaffinity to an antigen of interest at a neutral pH (e.g., pH of about7.0 to about 7.4) and enhanced dissociation of the antibody from anantigen-antigen-binding protein complex at a pH less than the neutral pH(e.g., at an endosomal pH, e.g. pH of about 5.5 to 6.0).

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 2 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 25° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin heavy chain locus as described herein has adissociative half-life (t_(1/2)) of less than 2 min at an acidic pH(e.g., pH of about 5.5 to about 6.0) at 37° C. In one embodiment, anantigen-binding protein comprising a heavy chain variable domainexpressed by the genetically modified immunoglobulin heavy chain locusas described herein has a dissociative half-life (t_(1/2)) of less than1 min at an acidic pH (e.g., pH of about 5.5 to about 6.0) at 25° C. Inone embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 1 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 37° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin locus as described herein has at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold, at least about 25-fold, or at least about 30-fold decrease indissociative half-life (t_(1/2)) at an acidic pH (e.g., pH of about 5.5to about 6.0) as compared to the dissociative half-life (t_(1/2)) of theantigen-binding protein at a neutral pH (e.g., pH of about 7.0 to about7.4).

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinlocus as described herein is characterized by improved pH-dependentrecyclability, enhanced serum half-life, or both as compared with awild-type antigen-binding protein without the genetic modification.

In one embodiment, the genetically modified immunoglobulin locusdescribed herein comprises a an enriched B cell population that, uponstimulation with an antigen of interest, is capable of producingantigen-binding proteins, e.g., antibodies, comprising a heavy chainvariable domain comprising one or more histidine residues. Theantigen-binding proteins as described herein when administered into asubject, exhibits an increased serum half-life over a correspondingwild-type antigen-binding protein, which possesses a similar orsufficiently similar amino acid sequence that encodes the heavy chainvariable domain but does not comprise a histidine residue in the heavychain variable domain. In some embodiments, the antigen-binding proteindescribed herein exhibits an increased serum half-life that is at leastabout 2-fold, at least about 5-fold, at least about 10-fold, at leastabout 15-fold, at least about 20-fold higher than the correspondingwild-type antigen-binding protein, which possesses a similar orsufficiently similar amino acid sequence that encodes the heavy chainvariable domain but does not comprise a histidine residue in the heavychain variable domain.

In one embodiment, the antigen-binding protein comprises animmunoglobulin heavy chain variable domain that is capable ofspecifically binding an antigen of interest with an affinity (K_(D))lower than 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, and 10⁻¹² at a neutralpH (e.g., pH of about 7.0 to about 7.4).

In one aspect, a method for obtaining an antigen-binding protein withenhanced recyclability and/or improved serum half-life is provided,comprising:

(a) immunizing a non-human animal having a genetically modifiedimmunoglobulin locus as described herein wherein the non-human animalcomprises an unrearranged human heavy chain variable region nucleotidesequence comprising an addition of least one histidine codon or asubstitution of at least one endogenous non-histidine codon with ahistidine codon;

(b) allowing the non-human animal to mount an immune response;

(c) harvesting a lymphocyte (e.g., a B cell) from the immunizednon-human animal;

(d) fusing the lymphocyte with a myeloma cell to form a hybridoma cell,and

(e) obtaining an antigen-binding protein produced by the hybridoma cell,wherein the antigen-binding protein exhibits enhanced recyclabilityand/or serum stability.

In one aspect, a genetically modified immunoglobulin heavy chain locusobtainable by any of the methods as described herein is provided.

In one aspect, a genetically modified non-human animal obtainable by anyof the methods as described herein is provided.

In various embodiments, the non-human animal is a mammal. In oneembodiment, the mammal is a rodent, e.g., a mouse, a rat, or a hamster.

In various embodiments, the genetically modified immunoglobulin loci asdescribed herein are present in the germline genome of a non-humananimal, e.g., a mammal, e.g., a rodent, e.g., a mouse, a rat, or ahamster.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate the amino acid sequences encoded by the threereading frames (i.e., stop, hydrophilic, and hydrophobic reading frames)of human D gene segments (D) and the amino acid sequences encoded by thethree reading frames of histidine-substituted human D gene segments(HD). Introduction of histidine codons (typed in bold) in thehydrophilic reading frame also changed many stop codons in the stopreading frame to Ser codons (typed in bold) but introduced few changesin the hydrophobic reading frame. The “*” symbol represents a stopcodon, and the comma between the two SEQ ID NOs indicates that there aretwo amino acid sequences separated by the stop codon.

FIG. 2 illustrates schemes for targeting pLMa0174 containing aspectinomycin selection cassette into the 5′ end of MAID 1116 (Step 1.BHR (Spec)). In Step 1, a chloramphenicol selection cassette, a neomycinselection cassette, a loxP site, two V_(H) gene segments (hV_(H)1-3 andhV_(H)1-2), the human Adam6 gene, all of which are located upstream ofhV_(H)6-1, were deleted from the clone and replaced by a spectinomycincassette to yield the VI433 clone. In Step 2 (BHR (Hyg +Spec)), pNTu0002containing a hygromycin cassette flanked by FRT sites was targeted intoa region comprising human immunoglobulin D gene segments. Via Step 2,all human D gene segments were deleted from VI433 and replaced with thehygromycin cassette to yield MAID6011 VI 434 (clone 1).

FIG. 3 illustrates schemes for assembling histidine-substituted human Dgene segments via sequential ligation.

FIG. 4 illustrates the introduction of pre-assembled,histidine-substituted human D gene segments containing a neomycincassette into a region between the most D-proximal V_(H) gene segment(V_(H) 6-1) and the most D-proximal J_(H) gene segment (J_(H)1) viaenzyme-mediated digestion (PI-SceI and I-CeuI) and ligation. Thisprocess removes the hygromycin cassette from MAID 6011 VI434 andintroduces pre-assembled human histidine-substituted D gene segmentsinto the clone. Bacterial cells comprising a successfully targeted cloneare selected based on both neomycin and spectinomycin resistance. Theresulting clone (MAID6012 VI469) comprises, from 5′ to 3′, (1) aspectinomycin selection cassette, (2) a 50 kb arm comprising a humanV_(H) gene segment (V_(H) 6-1), (3) a neomycin cassette flanked by loxPsites, (4) human D gene segments containing histidine substitutions (HD1.1-6.6 (9586 bp; SEQ ID NO: 1), HD 1.7-6.13 (9268 bp; SEQ ID NO: 2), HD1.14-6.19 (9441 bp; SEQ ID NO: 3), and HD 1.20-6.25, 1.26 (11592 bp; SEQID NO: 4)), (5) about 25 kb of a genomic region containing human J_(H)gene segments, (6) a mouse E_(i) sequence (SEQ ID NO: 5; an intronicenhancer that promotes V_(H) to DJ_(H) rearrangement in developing Bcells), and (7) a mouse IgM constant region nucleotide sequence (mlgMexon 1; SEQ ID NO: 7).

FIG. 5 illustrates schemes for deleting the human immunoglobulin heavychain D gene region from the MAID 1460 heterozygous ES cells bytargeting the 129 strain-derived chromosome of MAID 1460 het with thehygromycin selection cassette in MAID 6011 VI434.

FIG. 6 shows a list of primers and probes used to confirm a loss ofallele (LOA), a gain of allele (GOA), or a parental allele (Parental) inthe screening assays for identifying MAID 6011.

FIG. 7 illustrates schemes for constructing MAID 6012 het by targetingMAID 6011 heterozygous ES cells with MAID 6012 VI469. Electroporation ofthe MAID 6012 VI469 construct into the MAID 6011 heterozygous ES cellsyielded MAID 6012 heterozygous ES cells in which the 129 strain-derivedchromosome is modified to contain, from 5′ to 3′ direction, an FRT site,human V_(H) gene segments, a mouse genomic region comprising adam6genes, a floxed neomycin selection cassette, human D gene segmentscomprising histidine substitutions (HD 1.1-6.6 (9586 bp; SEQ ID NO: 1),HD 1.7-6.13 (9268 bp; SEQ ID NO: 2), HD 1.14-6.19 (9441 bp; SEQ ID NO:3), and HD 1.20-6.25, 1.26 (11592 bp; SEQ ID NO: 4)), human J_(H) genesegments, a mouse E_(i) sequence (SEQ ID NO: 5; an intronic enhancerthat promotes V_(H) to DJ_(H) rearrangement in developing B cells), anda mouse IgM constant region nucleotide sequence (mlgM exon 1; SEQ ID NO:7).

FIG. 8 shows a list of primers and probes used to confirm a loss ofallele (LOA), a gain of allele (GOA), or a parental allele (Parental) inthe screening assay for identifying MAID 6012.

FIG. 9 illustrates schemes for removing a neomycin cassette from MAID6012 heterozygous ES cells. Electroporation of a Cre-expressing plasmidinto the MAID 6012 ES cells lead to recombination and deletion of thefloxed neomycin cassette, yielding MAID 6013 heterozygous ES cells.

FIGS. 10A-10E illustrate human D gene segment nucleotide sequences withtranslations for each of the six reading frames, i.e., three readingframes for direct 5′ to 3′ orientation and three reading frames forinverted orientation (3′ to 5′ orientation). The “*” symbol represents astop codon, and the comma between two SEQ ID NOs indicates that thereare two amino acid sequences separated by the stop codon.

FIGS. 11-13 illustrate mRNA sequences and their encoded proteinsequences expressed by 6013 FO heterozygous mice, which comprisehistidine-substituted human D gene segments (HD 1.1-6.6 (9586 bp; SEQ IDNO: 1), HD 1.7-6.13 (9268 bp; SEQ ID NO: 2), HD 1.14-6.19 (9441 bp; SEQID NO: 3), and HD 1.20-6.25, 1.26 (11592bp; SEQ ID NO: 4)) in theimmunoglobulin heavy chain locus in their 129 strain-derived chromosome.The boxed sequences in each figure indicate the presence of histidinecodons in the CDR3 sequences derived from the genetically modifiedimmunoglobulin heavy chain locus comprising the histidine-substitutedhuman D gene segments. FWR represents frame region and CDR representscomplementarity determining region. In the alignment, the dot “.”indicates a sequence identical to the query sequence, and the dash “-”indicates a gap in the sequence.

FIG. 14 illustrates histidine incorporation frequency in immunoglobulinheavy chain CDR3 sequences. The X-axis represents the number ofhistidine codons appeared in each CDR3 sequence, and the Y-axisrepresents the corresponding proportion of reads. The “6013 F0 het”indicates CDR3 sequences expressed by the 6013 heterozygous micecomprising histidine-substituted D gene segments. The “VI3-Adam6”indicates CDR3 sequences obtained from control mice comprising humanV_(H), D, and J_(H) gene segments without the histidine modification asdescribed herein. The “ASAP” indicates CDR3 sequences obtained from theRegeneron antibody database, which was used as another control.

DETAILED DESCRIPTION OF THE INVENTION

This invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention is defined bythe claims.

Unless defined otherwise, all terms and phrases used herein include themeanings that the terms and phrases have attained in the art, unless thecontrary is clearly indicated or clearly apparent from the context inwhich the term or phrase is used. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, particular methods andmaterials are now described. All publications mentioned are herebyincorporated by reference

Definitions

The term “complementary determining region” or “CDR,” as used herein,includes an amino acid sequence encoded by a nucleic acid sequence of anorganism's immunoglobulin genes that normally (i.e., in a wild typeanimal) appears between two framework regions in a variable region of alight or a heavy chain of an immunoglobulin molecule (e.g., an antibodyor a T cell receptor). A CDR can be encoded by, for example, a germlinesequence or a rearranged sequence, and, for example, by a naïve or amature B cell or a T cell. A CDR can be somatically mutated (e.g., varyfrom a sequence encoded in an animal's germline), humanized, and/ormodified with amino acid substitutions, additions, or deletions. In somecircumstances (e.g., for a CDR3), CDRs can be encoded by two or moresequences (e.g., germline sequences) that are not contiguous (e.g., inan unrearranged nucleic acid sequence) but are contiguous in a B cellnucleic acid sequence, e.g., as a result of splicing or connecting thesequences (e.g., V-D-J recombination to form a heavy chain CDR3).

The term “dissociative half-life” or 1₁₂″ as used herein refers to thevalue calculated by the following formula: t_(1/2) (min)=(In2/k_(d))/60,wherein k_(d) represents a dissociation rate constant.

The term “germline” in reference to an immunoglobulin nucleic acidsequence includes a nucleic acid sequence that can be passed to progeny.

The phrase “heavy chain,” or “immunoglobulin heavy chain” includes animmunoglobulin heavy chain sequence, including immunoglobulin heavychain constant region sequence, from any organism. Heavy chain variabledomains include three heavy chain CDRs and four FR regions, unlessotherwise specified. Fragments of heavy chains include CDRs, CDRs andFRs, and combinations thereof. A typical heavy chain has, following thevariable domain (from N-terminal to C-terminal), a C_(H)1 domain, ahinge, a C_(H)2 domain, and a C_(H)3 domain. A functional fragment of aheavy chain includes a fragment that is capable of specificallyrecognizing an epitope (e.g., recognizing the epitope with a K_(D) inthe micromolar, nanomolar, or picomolar range), that is capable ofexpressing and secreting from a cell, and that comprises at least oneCDR. Heavy chain variable domains are encoded by variable regionnucleotide sequence, which generally comprises V_(H), D_(H), and J_(H)segments derived from a repertoire of V_(H), D_(H), and J_(H) segmentspresent in the germline. Sequences, locations and nomenclature for V, D,and J heavy chain segments for various organisms can be found in IMGTdatabase, which is accessible via the internet on the world wide web(www) at the URL “imgt.org.”

The phrase “light chain” includes an immunoglobulin light chain sequencefrom any organism, and unless otherwise specified includes human kappa(K) and lambda (A) light chains and a VpreB, as well as surrogate lightchains. Light chain variable domains typically include three light chainCDRs and four framework (FR) regions, unless otherwise specified.Generally, a full-length light chain includes, from amino terminus tocarboxyl terminus, a variable domain that includesFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant region aminoacid sequence. Light chain variable domains are encoded by the lightchain variable region nucleotide sequence, which generally compriseslight chain Wand light chain J_(L) gene segments, derived from arepertoire of light chain V and J gene segments present in the germline.Sequences, locations and nomenclature for light chain V and J genesegments for various organisms can be found in IMGT database, which isaccessible via the internet on the world wide web (www) at the URL“imgt.org.” Light chains include those, e.g., that do not selectivelybind either a first or a second epitope selectively bound by theepitope-binding protein in which they appear. Light chains also includethose that bind and recognize, or assist the heavy chain with bindingand recognizing, one or more epitopes selectively bound by theepitope-binding protein in which they appear.

The phrase “operably linked” refers to a relationship wherein thecomponents operably linked function in their intended manner. In oneinstance, a nucleic acid sequence encoding a protein may be operablylinked to regulatory sequences (e.g., promoter, enhancer, silencersequence, etc.) so as to retain proper transcriptional regulation. Inone instance, a nucleic acid sequence of an immunoglobulin variableregion (or V(D)J segments) may be operably linked to a nucleic acidsequence of an immunoglobulin constant region so as to allow properrecombination between the sequences into an immunoglobulin heavy orlight chain sequence.

The phrase “somatically mutated,” as used herein, includes reference toa nucleic acid sequence from a B cell that has undergoneclass-switching, wherein the nucleic acid sequence of an immunoglobulinvariable region, e.g., a heavy chain variable region (e.g., a heavychain variable domain or including a heavy chain CDR or FR sequence) inthe class-switched B cell is not identical to the nucleic acid sequencein the B cell prior to class-switching, such as, for example adifference in a CDR or a framework nucleic acid sequence between a Bcell that has not undergone class-switching and a B cell that hasundergone class-switching. The phrase “somatically mutated” includesreference to nucleic acid sequences from affinity-matured B cells thatare not identical to corresponding immunoglobulin variable regionnucleotide sequences in B cells that are not affinity-matured (i.e.,sequences in the genome of germline cells). The phrase “somaticallymatured” also includes reference to an immunoglobulin variable regionnucleic acid sequence from a B cell after exposure of the B cell to anepitope of interest, wherein the nucleic acid sequence differs from thecorresponding nucleic acid sequence prior to exposure of the B cell tothe epitope of interest. The term “somatically mutated” also refers tosequences from antibodies that have been generated in an animal, e.g., amouse having human immunoglobulin variable region nucleic acidsequences, in response to an immunogen challenge, and that result fromthe selection processes inherently operative in such an animal.

Non-Human Animals That Express Immunoglobulin Heavy Chain VariableDomain Comprising Histidine Residues

The described invention provides genetically modified non-human animalsthat can produce antigen-binding proteins with pH-dependent antigenbinding characteristics. In various embodiments, the antigen-bindingproteins produced by the genetically modified non-human animals asdescribed herein exhibit increased pH-dependent recycling efficiencyand/or enhanced serum half-life. In particular, the described inventionemploys genetic modifications in the immunoglobulin heavy chain locus tointroduce histidine codons into a human heavy chain variable regionnucleotide sequence and, optionally, to introduce a mutation(s) in aconstant region nucleotide sequence that encodes C_(H)2 and/or C_(H)3domains that increases the binding of the antibody constant region to anFcRn receptor, which facilitates recycling of the antigen-bindingprotein. Antigen-binding proteins comprising the modification may moreloosely bind its target in an acidic intracellular compartment (e.g., inan endosome where pH ranges from about 5.5 to about 6.0) than in anextracellular environment or at the surface of a cell (i.e., at aphysiological pH, e.g., a pH ranging from about 7.0 to about 7.4) due toprotonated histidine residues located in the antigen binding sites.Therefore, the antigen-biding proteins comprising the geneticmodifications as described herein would be able to be recycled morerapidly or efficiently than wild-type antigen-binding proteins that donot comprise such genetic modifications following target-mediatedendocytosis. Furthermore, since the modified histidine residues areprotonated only in an acidic environment, but not at a neutral pH, it isexpected that such modification would not affect binding affinity and/orspecificity of the antigen-binding protein toward an antigen of interestat a physiological pH.

In various aspects, non-human animals are provided comprisingimmunoglobulin heavy chain loci that comprise an unrearranged humanheavy chain variable region nucleotide sequence, wherein theunrearranged human heavy chain variable region nucleotide sequencecomprises an addition of least one histidine codon or a substitution ofat least one endogenous non-histidine codon with a histidine codon.

In various aspects, methods of making and using the non-human animalsare also provided. When immunized with an antigen of interest, thegenetically modified non-human animals are capable of generating B cellpopulations that produce antigen-binding proteins comprising heavy chainvariable domains with histidine residues, wherein the antigen-bindingproteins exhibit enhanced pH-dependent recycling and/or increased serumhalf-life. In various embodiments, the non-human animals generate B cellpopulations that express human heavy chain variable domains along withcognate human light chain variable domains. In various embodiments, thegenetically modified immunoglobulin heavy chain loci are present in agermline genome of the non-human animal.

In various embodiments, the genetically modified immunoglobulin heavychain locus comprises a modification that deletes or renders, all orsubstantially all, non-functional endogenous V_(H), D, and J_(H) genesegments; and the genetically modified locus comprises an unrearrangedheavy chain variable region nucleotide sequence comprising one or morehuman V_(H), D, and/or J_(H) gene segments having one or more histidinecodons, wherein the unrearranged heavy chain variable region nucleotidesequence is present at an endogenous location (i.e., where thenucleotide sequence is located in a wild-type non-human animal) orpresent ectopically (e.g., at a locus different from the endogenousimmunoglobulin chain locus in its genome, or within its endogenouslocus, e.g., within an immunoglobulin variable locus, wherein theendogenous locus is placed or moved to a different location in thegenome). In one embodiment, e.g., about 80% or more, about 85% or more,about 90% or more, about 95% or more, about 96% or more, about 97% ormore, about 98% or more, or about 99% or more of all endogenous heavychain V, D, or J gene segments are deleted or rendered non-functional.In one embodiment, e.g., at least 95%, 96%, 97%, 98%, or 99% ofendogenous functional heavy chain V, D, or J gene segments are deletedor rendered non-functional.

In one embodiment, the non-human animal is a mammal. Althoughembodiments directed to introducing histidine codons into anunrearranged human heavy chain variable gene sequence in a mouse areextensively discussed herein, other non-human animals are also providedthat comprise a genetically modified immunoglobulin locus containing anunrearranged human heavy chain variable region nucleotide sequencecomprising an addition of least one histidine codon or a substitution ofat least one endogenous non-histidine codon with a histidine codon. Suchnon-human animals include any of those which can be genetically modifiedto express the histidine-containing heavy chain variable domain asdisclosed herein, including, e.g., mouse, rat, rabbit, pig, bovine(e.g., cow, bull, buffalo), deer, sheep, goat, chicken, cat, dog,ferret, primate (e.g., marmoset, rhesus monkey), etc. For example, forthose non-human animals for which suitable genetically modifiable EScells are not readily available, other methods are employed to make anon-human animal comprising the genetic modification. Such methodsinclude, e.g., modifying a non-ES cell genome (e.g., a fibroblast or aninduced pluripotent cell) and employing somatic cell nuclear transfer(SCNT) to transfer the genetically modified genome to a suitable cell,e.g., an enucleated oocyte, and gestating the modified cell (e.g., themodified oocyte) in a non-human animal under suitable conditions to forman embryo. Methods for modifying a non-human animal genome (e.g., a pig,cow, rodent, chicken, etc. genome) include, e.g., employing a zincfinger nuclease (ZFN) or a transcription activator-like effectornuclease (TALEN) to modify a genome to include a nucleotides sequencethat encodes

In one embodiment, the non-human animal is a small mammal, e.g., of thesuperfamily Dipodoidea or Muroidea. In one embodiment, the geneticallymodified animal is a rodent. In one embodiment, the rodent is selectedfrom a mouse, a rat, and a hamster. In one embodiment, the rodent isselected from the superfamily Muroidea. In one embodiment, thegenetically modified animal is from a family selected from Calomyscidae(e.g., mouse-like hamsters), Cricetidae (e.g., hamster, New World ratsand mice, voles), Muridae (true mice and rats, gerbils, spiny mice,crested rats), Nesomyidae (climbing mice, rock mice, with-tailed rats,Malagasy rats and mice), Platacanthomyidae (e.g., spiny dormice), andSpalacidae (e.g., mole rates, bamboo rats, and zokors). In a specificembodiment, the genetically modified rodent is selected from a truemouse or rat (family Muridae), a gerbil, a spiny mouse, and a crestedrat. In one embodiment, the genetically modified mouse is from a memberof the family Muridae. In one embodiment, the animal is a rodent. In aspecific embodiment, the rodent is selected from a mouse and a rat. Inone embodiment, the non-human animal is a mouse.

In one embodiment, the non-human animal is a rodent that is a mouse of aC57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN,C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6N, C57BL/6NJ, C57BL/10,C57BL/10ScSn, C57BL/10Cr, and C57BL/OIa. In another embodiment, themouse is a 129 strain. In one embodiment, the 129 strain is selectedfrom the group consisting of 129P1, 129P2, 129P3, 129X1, 129S1 (e.g.,129S1/SV, 129S1/Svlm), 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6(129/SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, e.g., Festing et al.(1999) Revised nomenclature for strain 129 mice, Mammalian Genome10:836, see also, Auerbach et al. (2000) Establishment and ChimeraAnalysis of 129/SvEv- and C57BL/6-Derived Mouse Embryonic Stem CellLines). In one embodiment, the genetically modified mouse is a mix of anaforementioned 129 strain and an aforementioned C57BL strain (e.g., aC57BL/6 strain). In another embodiment, the mouse is a mix ofaforementioned 129 strains, or a mix of aforementioned C57BL/6 strains.In one embodiment, the 129 strain of the mix is a 129S6 (129/SvEvTac)strain. In another embodiment, the mouse is a mix of a 129/SvEv- and aC57BL/6-derived strain. In a specific embodiment, the mouse is a mix ofa 129/SvEv- and a C57BL/6-derived strain as described in Auerbach et al.2000 Bio Techniques 29:1024-1032. In another embodiment, the mouse is aBALB strain, e.g., BALB/c strain. In another embodiment, the mouse is amix of a BALB strain (e.g., BALB/c strain) and another aforementionedstrain.

In one embodiment, the non-human animal is a rat. In one embodiment, therat is selected from a Wistar rat, an LEA strain, a Sprague Dawleystrain, a Fischer strain, F344, F6, and Dark Agouti. In one embodiment,the rat strain is a mix of two or more of a strain selected from thegroup consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, andDark Agouti.

In one embodiment, the non-human animal is a mouse. In one embodiment,the mouse is a VELOCIMMUNE® humanized mouse.

VELOCIMMUNE® humanized mice (see, e.g., U.S. Pat. No. 6,596,541, U.S.Pat. No. 7,105,348, and

US20120322108A1, which are incorporated herein by reference in theirentireties), which contain a precise replacement of mouse immunoglobulinvariable regions with human immunoglobulin variable regions at theendogenous mouse loci, display a surprising and remarkable similarity towild-type mice with respect to B cell development. VELOCIMMUNE®humanized mice display an essentially normal, wild-type response toimmunization that differed only in one significant respect fromwild-type mice—the variable regions generated in response toimmunization are fully human.

VELOCIMMUNE® humanized mice contain a precise, large-scale replacementof germline variable region nucleotide sequences of mouse immunoglobulinheavy chain (IgH) and immunoglobulin light chain (e.g., κ light chain,Igκ) with corresponding human immunoglobulin variable region nucleotidesequences, at the endogenous loci (see, e.g., U.S. Pat. No. 6,596,541,U.S. Pat. No. 7,105,348, US 20120322108A1, which are incorporated hereinby reference in their entireties). In total, about six megabases ofmouse loci are replaced with about 1.5 megabases of human genomicsequence. This precise replacement results in a mouse with hybridimmunoglobulin loci that make heavy and light chains that have a humanvariable regions and a mouse constant region. The precise replacement ofmouse V_(H)-D-J_(H) and Vκ-Jκ segments leave flanking mouse sequencesintact and functional at the hybrid immunoglobulin loci. The humoralimmune system of the mouse functions like that of a wild-type mouse. Bcell development is unhindered in any significant respect and a richdiversity of human variable regions is generated in the mouse uponantigen challenge.

VELOCIMMUNE® humanized mice are possible because immunoglobulin genesegments for heavy and κ light chains rearrange similarly in humans andmice, which is not to say that their loci are the same or even nearlyso—clearly they are not. However, the loci are similar enough thathumanization of the heavy chain variable gene locus can be accomplishedby replacing about three million base pairs of contiguous mouse sequencethat contains all the V_(H), D, and J_(H) gene segments with about onemillion bases of contiguous human genomic sequence covering basicallythe equivalent sequence from a human immunoglobulin locus.

In some embodiments, further replacement of certain mouse constantregion nucleotide sequences with human constant region nucleotidesequences (e.g., replacement of mouse heavy chain C_(H)1 nucleotidesequence with human heavy chain C_(H)1 nucleotide sequence, andreplacement of mouse light chain constant region nucleotide sequencewith human light chain constant region nucleotide sequence) results inmice with hybrid immunoglobulin loci that make antibodies that havehuman variable regions and partly human constant regions, suitable for,e.g., making fully human antibody fragments, e.g., fully human Fab's.Mice with hybrid immunoglobulin loci exhibit normal variable genesegment rearrangement, normal somatic hypermutation frequencies, andnormal class switching. These mice exhibit a humoral immune system thatis indistinguishable from wild type mice, and display normal cellpopulations at all stages of B cell development and normal lymphoidorgan structures—even where the mice lack a full repertoire of humanvariable region nucleotide segments. Immunizing these mice results inrobust humoral responses that display a wide diversity of variable genesegment usage.

The precise replacement of the mouse germline variable region nucleotidesequence allows for making mice that have partly human immunoglobulinloci. Because the partly human immunoglobulin loci rearrange,hypermutate, and class switch normally, the partly human immunoglobulinloci generate antibodies in a mouse that comprise human variableregions. Nucleotide sequences that encode the variable regions can beidentified and cloned, then fused (e.g., in an in vitro system) with anysequences of choice, e.g., any immunoglobulin isotype suitable for aparticular use, resulting in an antibody or antigen-binding proteinderived wholly from human sequences.

In various embodiments, at least one histidine codon is present in anunrearranged heavy chain variable region nucleotide sequence thatencodes an N-terminal region, a loop 4 region, a CDR1, a CDR2, a CDR3,or a combination thereof.

In various embodiments, at least one histidine codon is present in anunrearranged heavy chain variable region nucleotide sequence thatencodes a framework region (FR) selected from the group consisting ofFR1, FR2, FR3, and FR4.

In various aspects, the genetically modified immunoglobulin locuscomprises a nucleotide sequence wherein at least one codon has beenreplaced with a histidine codon.

In various aspects, the genetically modified immunoglobulin locuscomprises an unrearranged human heavy chain variable region nucleotidesequence comprising a substitution of at least one endogenousnon-histidine codon with a histidine codon.

In one embodiment, 2 or more, 3 or more, 4 or more, 5 or more, 6 ormore, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 ormore, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 ormore, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 ormore, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 ormore, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 ormore, 37 or more, 38 or more, 39 or more, 40 or more, 41 or more, 42 ormore, 43 or more, 44 or more, 45 or more, 46 or more, 47 or more, 48 ormore, 49 or more, 50 or more, 51 or more, 52 or more, 53 or more, 54 ormore, 55 or more, 56 or more, 57 or more, 58 or more, 59 or more, 60 ormore, or 61 or more of the endogenous non-histidine codons are replacedwith histidine codons.

Previous studies on reading frame usage of human immunoglobulin D genesegments have shown that, of the three reading frames (i.e., stop,hydrophobic, and hydrophilic), the stop frame is used very infrequently.Apparently, some stop frames are chewed back and result in expression.However, stop reading frames are used at such a low frequency that forthe purposes of engineering histidine codons, it is more efficient notto use the stop reading frame. As between hydrophilic and hydrophobicreading frames, the hydrophilic reading frame appears to be preferred.Thus, in one embodiment, the hydrophilic reading frame of human D genesegments is engineered to contain one or more histidine codons (ascompared with the stop frame or with the hydrophobic frame).

Methods of introducing a mutation in vitro, e.g., site-directedmutagenesis, are well known in the art. In some embodiments of thedescribed invention, histidine codons are enriched by designinghistidine-substituted human D gene segments in silico (e.g., mutation ofY, D, and N codons to H codons, e.g., CAT, CAC), which are synthesized(e.g., chemical synthesis) with (unique) restriction enzyme sites forligating them back together. The synthesized D gene segments are madewith the appropriate recombination signal sequences (RSS) upstream anddownstream. In one embodiment, when ligated to one another, thesynthesized histidine-substituted D gene segments include the intergenicsequences observed in a human between each D gene segment.

It is understood that the codons that encode the one or more histidines,upon rearrangement and/or somatic hypermutation, may change such thatone or more of the histidines will be changed to another amino acid.However, this may not occur for each and every codon encoding histidine,in each and every rearrangement in the non-human animal. If such changesoccur, the changes may occur in some but not all B cells or in some butnot all heavy chain variable sequences.

In various aspects, the genetically modified immunoglobulin locuscomprises a human heavy chain V, D, and J gene segment, wherein at leastone of the human D gene segment has been inverted 5′ to 3′ with respectto a corresponding wild-type sequence, and wherein at least one readingframe of the inverted human D gene segment comprises a histidine codon.

In various embodiments, the nucleotide sequence comprises one or more, 2or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 ormore, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 ormore, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 ormore, 21 or more, 22 or more, 23 or more, 24 or more, or 25 or more ofhistidine codons.

There are 25 functional human D gene segments in 6 families of 3-5members each (one family—the D7 family—has a single member). Directrecombination of human D gene segments is much more frequent thaninversion, although inverted reading frames exhibit more histidinecodons. Certain D gene segments and reading frames are used morefrequently than others. All three direct reading frames and all threeinverted orientation reading frames for all the functional D genesegments are presented in FIGS. 10A-10E. As shown in FIGS. 10A-10E,there are many more histidine codons in inverted reading frames than indirect reading frames. More specifically, there are 34 histidines ininverted reading frames and only four in direct reading frames. Inaddition, of the four in direct reading frames, three histidines areencoded by pseudogenes or present in alternate alleles. Therefore, thereis only a single direct reading frame of a germline human D gene segmentthat contains a histidine codon, with further histidine codons possiblyencountered in alternate alleles (presumably in subsets of the humanpopulation).

Inverted D rearrangements are extremely rare. Tuaillon et al. (J.Immunol., 154(12): 5453-6465, incorporated by reference herein in itsentirety) showed that usage of inverted reading frames (as measured bylimiting dilution PCT) is very rare, i.e., that the ratio of direct toindirect rearrangements are, in most cases, 100 to 1000. To the extentthat the ratio of direct to indirect rearrangement was low, it was onlyobserved in those D segments that exhibit very low usage. It was alsoshown that D gene segment family 7, which is located adjacent to J1 (fardown from other D family members) is mostly used in fetuses, butexhibits a low usage in adults (Schroeder et al., Immunology 30, 2006,119-135, incorporated by reference herein in its entirety). Therefore,in one embodiment, D family 7 sequences are not inverted 5′ to 3′.

In one embodiment, at least two, at least three, at least four, at leastfive, at least six, at least seven, at least eight, at least nine, atleast ten, at least eleven, at least twelve, at least thirteen, at leastfourteen, at least fifteen, at least sixteen, at least seventeen, atleast eighteen, at least nineteen, at least twenty, at least twenty one,at least twenty two, at least twenty three, at least twenty four, or allor substantially all of the human functional D gene segments areinverted 5′ to 3′ with respect to corresponding wild type sequences.

In one embodiment, the human immunoglobulin heavy chain variable domaincomprising at least one non-naturally occurring histidine residueexhibits pH-dependent antigen binding characteristics. For example, anantibody comprising the modified immunoglobulin heavy chain variabledomain binds a target with sufficient affinity at around a neutral pH(e.g., pH of about 7.0 to about 7.4), but either does not bind or bindsweaker to the same target at an acidic pH (e.g., pH of about 5.5 toabout 6.0). In one embodiment, the acidic pH is selected from about 5.5,about 5.6, about 5.7, about 5.8, about 5.9, and about 6.0. In oneembodiment, the neutral pH is selected from about 7.0, about 7.1, about7.2, about 7.3, and about 7.4.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 2 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 25° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin heavy chain locus as described herein has adissociative half-life (t_(1/2)) of less than 2 min at an acidic pH(e.g., pH of about 5.5 to about 6.0) at 37° C. In one embodiment, anantigen-binding protein comprising a heavy chain variable domainexpressed by the genetically modified immunoglobulin heavy chain locusas described herein has a dissociative half-life (t_(1/2)) of less than1 min at an acidic pH (e.g., pH of about 5.5 to about 6.0) at 25° C. Inone embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 1 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 37° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin locus as described herein has at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold, at least about 25-fold, or at least about 30-fold decrease indissociative half-life (t_(1/2)) at an acidic pH (e.g., pH of about 5.5to about 6.0) as compared to the dissociative half-life (t_(1/2)) of theantigen-binding protein at a neutral pH (e.g., pH of about 7.0 to about7.4).

In one embodiment, antigen binding proteins comprising the geneticallymodified human immunoglobulin heavy chain variable domain is capable ofspecifically binding an antigen of interest with an affinity (K_(D))lower than 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ or 10⁻¹⁰, 10⁻¹¹, 10⁻¹² at a neutral orphysiological pH (pH of about 7.0 to about 7.4).

The altered binding property of the immunoglobulin heavy chain variabledomain at an acidic pH (e.g., pH of about 5.5 to about 6.0) would, insome circumstances, allow faster turnover of the antibody because thetherapeutic antibody will bind a target on a cell's surface, beinternalized into an endosome, and more readily or more rapidlydissociate from the target in the endosome, so that the therapeutic canbe recycled to bind yet another molecule of target present in anothercell. This would allow one to administer a therapeutic antibody at alower dose, or administer the therapeutic antibody less frequently. Thisis particularly useful in a situation where it is not desirable toadminister a therapeutic antibody frequently, or administer at a levelabove a certain dosage for safety or toxicity reasons.

In various embodiments, the human immunoglobulin heavy chain variableregion nucleotide sequence as described herein is operably linked to ahuman or non-human heavy chain constant region nucleotide sequence(e.g., a heavy chain constant region nucleotide sequence that encodes animmunoglobulin isotype selected from IgM, IgD, IgG, IgE, and IgA). Invarious embodiments, the human or non-human heavy chain constant regionnucleotide sequence is selected from the group consisting of a C_(H)1, ahinge, a C_(H2), a C_(H3), and a combination thereof. In one embodiment,the constant region nucleotide sequence comprises a C_(H)1, a hinge, aC_(H)2, and a C_(H)3 (e.g., C_(H)1-hinge-a C_(H)2-C_(H)3).

In various embodiments, the heavy chain constant region nucleotidesequence is present at an endogenous locus (i.e., where the nucleotidesequence is located in a wild-type non-human animal) or presentectopically (e.g., at a locus different from the endogenousimmunoglobulin chain locus in its genome, or within its endogenouslocus, e.g., within an immunoglobulin variable locus, wherein theendogenous locus is placed or moved to a different location in thegenome).

In one embodiment, the heavy chain constant region nucleotide sequencecomprises a modification in a C_(H)2 or a C_(H)3, wherein themodification increases the affinity of the heavy chain constant regionamino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

The neonatal Fc receptor for IgG (FcRn) has been well characterized inthe transfer of passive humoral immunity from a mother to her fetusacross the placenta and proximal small intestine (Roopenian, D. andAkilesh, S., Nat. Rev. Immun., 2007, 7:715-725, which is incorporated byreference herein in its entirety). FcRn binds to the Fc portion of IgGat a site that is distinct from the binding sites of the classical FcyRsor the C1q component of complement, which initiates the classicalpathway of complement activation. More specifically, it was shown thatFcRn binds the C_(H)2-C_(H)3 hinge region of IgG antibodies—a versatileregion of Fc that also binds Staphylococcal protein A, Streptococcalprotein G, and the rheumatoid factor. In contrast to other Fc-bindingproteins, however, FcRn binds the Fc region of IgG in a strictlypH-dependent manner; at physiological pH 7.4, FcRn does not bind IgG,whereas at the acidic pH of the endosome (e.g., where the pH ranges fromabout 5.5 to about 6.0), FcRn exhibits a low micromolar to nanomolaraffinity for the Fc region of IgG. This pH-dependent interaction hasbeen shown to be mediated by the titration of histidine residues in theC_(H)2-C_(H)3 region of IgG and their subsequent interaction with acidicresidue on the surface of FcRn (Roopenian, D. and Akilesh, S., Nat. Rev.Immun., 2007, 7:715-725, incorporated by reference in its entirety).

Various mutations in the C_(H)2-C_(H)3 region of IgG that can increasethe affinity of Fc region to FcRn at an acidic pH are known in the art.These include, but are not limited to, modification at position 250(e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T),254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or a modification at428 and/or 433 (e.g., L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or amodification at 250 and/or 428; or a modification at 307 or 308 (e.g.,308F, V308F), and 434. In another example, the modification can comprisea 428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I(e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K)and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 52Y,254T, and 256E) modification; a 250Q and 428L modification, or acombination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 252 and 257,wherein the modification increases the affinity of the human C_(H)2amino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 307 and 311,wherein the modification increases the affinity of the C_(H)2 amino acidsequence to FcRn in an acidic environment (e.g., in an endosome where pHranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)3 amino acid sequence, wherein the C_(H)3 aminoacid sequence comprises at least one modification between amino acidresidues at positions 433 and 436, wherein the modification increasesthe affinity of the C_(H)3 amino acid sequence to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0).

In one embodiment, the human constant region amino acid sequence encodedby the heavy chain constant region nucleotide sequence described hereincomprises a mutation selected from the group consisting of M428L, N434S,and a combination thereof. In one embodiment, the human constant regionamino acid sequence comprises a mutation selected from the groupconsisting of M428L, V259I, V308F, and a combination thereof. In oneembodiment, the human constant region amino acid sequence comprises anN434A mutation. In one embodiment, the human constant region amino acidsequence comprises a mutation selected from the group consisting ofM252Y, S254T, T256E, and a combination thereof. In one embodiment, thehuman constant region amino acid sequence comprises a mutation selectedfrom the group consisting of T250Q, M248L, or both. In one embodiment,the human constant region amino acid sequence comprises a mutationselected from the group consisting of H433K, N434Y, or both.

In one embodiment, the heavy chain constant region amino acid sequenceis a non-human constant region amino acid sequence, and the heavy chainconstant region amino acid sequence comprises one or more of any of thetypes of modifications described above.

In one embodiment, the heavy chain constant region nucleotide sequenceis a human heavy chain constant region amino acid sequence, and thehuman heavy chain constant region amino acid sequence comprises one ormore of any of the types of modifications described above.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

p Example 1 Construction of Humanized Immunoglobulin Heavy Chain LociComprising Histidine-Substituted D Gene Segments

Construction of immunoglobulin heavy chain loci comprisinghistidine-substituted human D gene segments was carried out by series ofhomologous recombination reactions in bacterial cells (BHR) usingBacterial Artificial Chromosome (BAC) DNA. Several targeting constructsfor creation of a genetically engineered mouse that expresses a heavychain variable domain comprising one or more histidine residues weregenerated using VELOCIGENE® genetic engineering technology (see, e.g.,U.S. Pat. No. 6,586,251 and Valenzuela, D. M. et al. (2003),High-throughput engineering of the mouse genome coupled withhigh-resolution expression analysis, Nature Biotechnology 21(6):652-659,which is incorporated herein by reference in their entireties).

Initially, human D gene segments were synthesized in silico as fourpieces (4 repeats) in which the codons encoding tyrosine (Y), asparagine(N), serine (S), glycine (G), and aspartate (D) in the hydrophilic framewere substituted with histidine codons (hereinafter“histidine-substituted human D gene segments”, i.e., HD 1.1-6.6 (9586bp; SEQ ID NO: 1), HD 1.7-6.13 (9268 bp; SEQ ID NO: 2), HD 1.14-6.19(9441 bp; SEQ ID NO: 3), and HD 1.20-6.25, 1.26 (11592 bp; SEQ ID NO: 4)(FIG. 3). The four repeats also contained unique restriction enzymesites at the ends for ligating them back together. The specific locationof the histidine substitutions (labeled in bold type) in each human Dgene segment is shown in FIGS. 1A and 1B in the column labeled“Hydrophilic.” As shown in FIG. 1, while the modification introducedhistidine codons in the hydrophilic reading frame, it also changed somestop codons to serine codons in the “Stop” reading frame. Themodification, however, made few changes in the “Hydrophobic” readingframe. The detailed procedure for ligating the four synthesized Dsegment repeats is illustrated in FIG. 3 (sequential ligation). Theresulting clone contained, from 5′ to 3′, a 5′ mouse homology arm, afloxed neomycin cassette, human D gene segments comprising histidinesubstitutions (i.e., HD 1.1-6.6 (9586 bp; SEQ ID NO: 1), HD 1.7-6.13(9268 bp; SEQ ID NO: 2), HD 1.14-6.19 (9441 bp; SEQ ID NO: 3), and HD1.20-6.25, 1.26 (11592 bp; SEQ ID NO: 4)), a chloramphenicol selectioncassette, and a 3′ homology arm.

The following six genetic modifications were carried out in order toreplace the endogenous human D gene segments in the VELOCIMMUNE®humanized mouse with the histidine-substituted human D gene segmentsdescribed above.

First, pLMa0174, containing a spectinomycin selection cassette and anAsiSI restriction site, was targeted into the 5′ end of the MAID 1116clone (Step 1. BHR (Spec); FIG. 2). During Step 1, a chloramphenicolselection cassette, a neomycin selection cassette, a loxP site, twoV_(H) gene segments (hV_(H)1-3 and hV_(H)1-2), and the human Adam6pgene, all of which are located 5′ upstream of hV_(H)6-1, were deletedfrom the MAID 1116 clone and replaced by a spectinomycin cassette toyield the VI433 clone.

Second, in Step 2 (BHR (Hyg+Spec); FIG. 2), pNTu0002 containing ahygromycin cassette flanked by FRT sites was targeted into a regioncomprising human immunoglobulin D_(H) gene segments. During Step 2, allhuman heavy chain D gene segments were deleted from VI433 and replacedwith the hygromycin cassette to yield MAID6011 VI434 (clone 1). Themodification also introduced the PI-SceI and the I-CeuI restrictionsites at the 5′ and 3′ end of the hygromycin cassette.

Third, the genomic region comprising histidine-substituted human D genesegments (HD 1.1-6.6 (9586 bp; SEQ ID NO: 1), HD 1.7-6.13 (9268 bp; SEQID NO: 2), HD 1.14-6.19 (9441 bp; SEQ ID NO: 3), and HD 1.20-6.25, 1.26(11592 bp; SEQ ID NO: 4)) were introduced into a region between thePI-SceI and the I-CeuI sites of MAID 6011 VI434 via restrictiondigestion and ligation (PI-SceI/I-CeuI Ligation modified 1116(Kan+Spec); FIG. 4). This yielded MAID6012 VI469 containing, from 5′ to3′, a spectinomycin cassette, about 50 kb of a genomic region comprisingV_(H)6-1, a floxed neomycin cassette, about 40 kb of thehistidine-substituted human D gene segments (HD 1.1-6.6 (9586 bp; SEQ IDNO: 1), HD 1.7-6.13 (9268 bp; SEQ ID NO: 2), HD 1.14-6.19 (9441 bp; SEQID NO: 3), and HD 1.20-6.25, 1.26 (11592 bp; SEQ ID NO: 4)), and about25 kb of a genomic region containing human J_(H) gene segments, followedby a mouse E_(i) (mIgH intronic enhancer; SEQ ID NO: 5), a mouse switchregion (SEQ ID NO: 6), and a mouse IgM constant region nucleotidesequence (mIgM exon 1; SEQ ID NO: 7). Bacterial cells containing themodification were selected based on Kanamycin and Spectinomycinselection.

Fourth, MAID 1460 heterozygous mouse ES cells were targeted with MAID6011 VI434 via electroporation in order to remove all endogenous human Dgene segments from the MAID 1460 clone as illustrated in FIG. 5. Thisyielded MAID 6011 heterozygous mouse ES cells comprising in itsimmunoglobulin heavy chain locus (at the 129 strain-derived chromosome),from 5′ to 3′, an FRT site, human V_(H) gene segments, a mouse genomicregion encompassing adam6a/b genes, a hygromycin cassette flanked by FRTsites, and human J_(H) segments, followed by a mouse E_(i) sequence andan IgM constant region nucleotide sequence. The genetic modification ofMAID 6011 (a loss of alleles, a gain of alleles, and presence ofparental alleles) was confirmed by using the probes and primers as shownin FIG. 6.

Fifth, MAID 6011 heterozygous mouse ES cells were electroporated withMAID 6012 VI469 in order to introduce histidine-substituted human D genesegments (i.e., HD 1.1-6.6 (9586 bp; SEQ ID NO: 1), HD 1.7-6.13 (9268bp; SEQ ID NO: 2), HD 1.14-6.19 (9441 bp; SEQ ID NO: 3), and HD1.20-6.25, 1.26 (11592 bp; SEQ ID NO: 4)) into MAID 6011. The targetingstep removed the floxed hygromycin selection cassette from MAID 6011 andreplaced the sequence with the histidine-substituted human D genesegments. This lead to MAID 6012 hetrozygous ES cells comprising awild-type C57BL/6 strain-derived chromosome and a genetically modified129 strain-derived chromosome comprising human wild-type V_(H) and J_(H)gene segments and the histidine-substituted human D gene segmentsdescribed herein. In addition, the ES cells contained a mouse genomicregion encompassing adam6a/b genes and a floxed neomycin cassettebetween the V_(H) and D segments (FIG. 7). The genetic modification ofMAID 6012 (a loss of alleles, a gain of alleles, and presence ofparental alleles) was confirmed by using the probes and primers as shownin FIG. 8.

Lastly, MAID 6012 ES cells were electroporated with a plasmid thatexpresses a Cre recombinase in order to remove the neomycin selectioncassette from the MAID 6012 ES cells, resulting in MAID 6013heterozygous ES cells (FIG. 9). The final MAID 6013 heterozygous (“MAID6013 het”) ES cell contains a wild-type C57BL/6 strain-derivedchromosome and a genetically modified, 129 strain-derived chromosomecomprising in its immunoglobulin heavy chain locus, from 5′ to 3′, (1)an FRT site; (2) human V_(H) gene segments; (3) a mouse genomic regionencompassing adam6a/b genes; (4) a floxed neomycin selection cassette;(5) histidine-substituted human D gene segments (HD 1.1-6.6 (9586 bp;SEQ ID NO: 1), HD 1.7-6.13 (9268 bp; SEQ ID NO: 2), HD 1.14-6.19 (9441bp; SEQ ID NO: 3), and HD 1.20-6.25, 1.26 (11592 bp; SEQ ID NO: 4)); (6)human J_(H) gene segments; followed by (7) a mouse E_(i) sequence (mIgHintronic enhancer; SEQ ID NO: 5), (8) a switch region (SEQ ID NO: 6);and (9) a mouse IgM constant region nucleotide sequence (mIgM exon 1;SEQ ID NO: 7) as illustrated in FIG. 9.

The targeted ES cells (MAID 6013) described above were used as donor EScells and introduced into an 8-cell stage mouse embryo by theVELOCIMOUSE® method (see, e.g., U.S. Pat. No. 7,576,259, U.S. Pat. No.7,659,442, U.S. Pat. No. 7,294,754, US 2008-0078000 A1, all of which areincorporated by reference herein in their entireties). Mice bearing thegenetically modified immunoglobulin heavy chain locus comprising thehistidine-substituted human heavy chain D gene segments described hereinwere identified by genotyping using the primers and probes set forth inFIG. 8. The resulting genetically modified FO mouse was crossed to awild-type mouse to obtain F1 offspring. F1 pups were genotyped, and theF1 pups that are heterozygous for the genetically modifiedimmunoglobulin locus comprising histidine-substituted human heavy chainD gene segments were selected for further characterization.

Example 2 Analysis of Rearranged Heavy Chain Variable Region NucleotideSequences

Next, it was examined whether the genetically modified mouse comprisinghistidine-substituted human D gene segments described herein, i.e., 6013F0 heterozygous mouse, which comprises in its germline a 129strain-derived chromosome comprising human V_(H), J_(H) gene segments,and histidine-substituted human D gene segments (HD 1.1-6.6 (9586 bp;SEQ ID NO: 1), HD 1.7-6.13 (9268 bp; SEQ ID NO: 2), HD 1.14-6.19 (9441bp; SEQ ID NO: 3), and HD 1.20-6.25, 1.26 (11592 bp; SEQ ID NO: 4), canexpress rearranged heavy chain V(D)J sequences comprising one or morehistidine codons derived from the genetically modified immunoglobulinheavy chain locus.

To this end, mRNA sequences encoding IgM heavy chain variable regionwere analyzed for the presence of IgM CDR3 sequences derived from thehistidine-substituted human D gene segments via high throughputsequencing. Briefly, spleens were harvested and homogenized in 1×PBS(Gibco) using glass slides. Cells were pelleted in a centrifuge (500×gfor 5 minutes), and red blood cells were lysed in ACK Lysis buffer(Gibco) for 3 minutes. Cells were washed with 1×PBS and filtered using a0.7 μm cell strainer. B-cells were isolated from spleen cells using MACSmagnetic positive selection for CD19 (Miltenyi Biotec). Total RNA wasisolated from pelleted B-cells using the RNeasy Plus kit(Qiagen).PolyA+mRNA was isolated from total RNA using the Oligotex® Direct mRNAmini kit (Qiagen).

Double-stranded cDNA was prepared from splenic B cell mRNA by 5′ RACEusing the SMARTer™ Pico cDNA Synthesis Kit (Clontech). The Clontechreverse transcriptase and dNTPs were substituted with Superscript II anddNTPs from Invitrogen. Heavy chain variable region (V_(H)) antibodyrepertoires were amplified from the cDNA using primers specific for IgMconstant regions and the SMARTer™ 5′ RACE primer (Table 1). PCR productswere cleaned up using a QIAquick® PCR Purification Kit (Qiagen). Asecond round of PCR was done using the same 5′ RACE primer and a nested3′ primer specific for the IgM constant regions (Table 2). The secondround PCR products were purified using a SizeSelect™ E-gel® system(Invitrogen). A third PCR was performed with primers that added 454adapters and barcodes. The third round PCR products were purified usingAgencourt® AMPure® XP Beads. Purified PCR products were quantified bySYBR®-qPCR using a KAPA Library Quantification Kit (KAPA Biosystems).Pooled libraries were subjected to emulsion PCR (emPCR) using the 454 GSJunior Titanium Series Lib-A emPCR Kit (Roche Diagnostics) andbidirectional sequencing using Roche 454 GS Junior instrument accordingto the manufacturer's protocols.

TABLE 1 NAME SEQUENCE 3′ mIgM CH1 outer TCTTATCAGACAGGGGGCTCTC (SEQ ID NO: 321)

TABLE 2  NAME 3′ mIgM  GGAAGACATTTGGGAAGGACTG (SEQID NO: 322) CH1 inner

Bioinfomatic Analysis

The 454 sequences were sorted based on the sample barcode perfect matchand trimmed for quality. Custom D database was created usinghistidine-substituted human D-gene segments. Sequences were annotatedbased on alignment of rearranged Ig sequences to human germline V and Jgene segments database using local installation of igblast (NCBI,v2.2.25+). Sequences derived from the endogenous mouse immunoglobulinheavy chain locus were filtered out using similarity threshold of 90%. Asequence was marked as ambiguous and removed from analysis when multiplebest hits with identical score were detected. A set of perl scripts wasdeveloped to analyze results and store data in mysql database. The CDR3region was defined between conserved C codon and FGXG motif (SEQ ID NO:323) for light chains and WGXG motif (SEQ ID NO: 324) for heavy chains.CDR3 length was determined using only productive antibodies. Number ofhistidine codons was calculated for each CDR3 region.

As shown in FIGS. 11-13, the 6013 F0 heterozygous mice expressed adiverse repertoire of rearranged heavy chain variable region mRNAsequences (rearranged V-D-J sequences) encoding one or more histidinecodons in CDR3. The sequencing and alignment data suggested that thehistidine codons appeared in CDR3 sequences were derived from varioushistidine-substituted human D gene segments present in the geneticallymodified immunoglobulin heavy chain locus of the 6013 mice describedherein. In addition, as compared with control mice comprising humanV_(H), D_(H), J_(H) gene segments and mouse adam6 genes (VI3-Adam6, USPublication No. 2012/0322108A1, which is incorporated by reference inits entirety), the genetically modified 6013 F0 heterozygous miceexhibited a higher frequency of histidine occurrence in the heavy chainCDR3 sequences (FIG. 14).

While the described invention has been described with reference to thespecific embodiments thereof it should be understood by those skilled inthe art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adopt aparticular situation, material, composition of matter, process, processstep or steps, to the objective spirit and scope of the describedinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A method of producing a nucleic acid encoding ahuman immunoglobulin heavy chain variable domain with at least onehistidine, comprising: obtaining from a lymphocyte of a non-humananimal, or a hybridoma produced from the lymphocyte, a nucleic acidcomprising a rearranged human immunoglobulin heavy chain variable regiongene sequence that encodes a human immunoglobulin heavy chain variabledomain, wherein the non-human animal comprises in its germline genome anunrearranged human immunoglobulin heavy chain variable region nucleotidesequence that comprises an addition of at least one histidine codon or asubstitution of at least one non-histidine codon with a histidine codon,wherein the added or substituted histidine codon is not encoded by acorresponding human germline heavy chain variable region gene segment,and wherein the added or substituted histidine codon is present in acomplementary determining region 3 (CDR3) encoding sequence.
 2. Themethod of claim 1, further comprising immunizing the non-human animalwith an antigen of interest, and allowing the animal to mount an immuneresponse to the antigen before obtaining the nucleic acid.
 3. The methodof claim 2, wherein the human immunoglobulin heavy chain variable domainspecifically binds the antigen of interest.
 4. The method of claim 3,wherein the obtained rearranged human immunoglobulin heavy chainvariable region gene sequence comprises at least one somatichypermutation.
 5. The method of claim 1, wherein the unrearranged humanimmunoglobulin heavy chain variable region nucleotide sequence isoperably linked to an endogenous non-human immunoglobulin heavy chainconstant region gene sequence at an endogenous non-human immunoglobulinheavy chain locus.
 6. The method of claim 1, wherein the non-humananimal further comprises in its germline genome an unrearranged humanimmunoglobulin light chain variable region nucleotide sequencecomprising unrearranged human V_(L) and unrearranged human J_(L) genesegments; and wherein the lymphocyte, or hybridoma produced therefrom,expresses a human immunoglobulin light chain variable domain that iscognate to the human immunoglobulin heavy chain variable domain.
 7. Themethod of claim 1, wherein the unrearranged human immunoglobulin heavychain variable region nucleotide sequence further comprises an additionof at least one histidine codon or a substitution of at least onenon-histidine codon with a histidine codon in a CDR1 encoding sequence,a CDR2 encoding sequence, an N terminal encoding sequence or a loopencoding sequence.
 8. The method of claim 1, wherein the CDR3 encodingsequence is selected from a human germline V_(H) gene segment sequence,a human germline D gene segment sequence, a human germline J_(H) genesegment sequence, and a combination thereof.
 9. The method of claim 1,wherein the non-histidine codon that is substituted with the histidinecodon encodes the amino acid selected from the group consisting of Y, N,D, Q, S, W, and R.
 10. The method of claim 1, wherein the added orsubstituted histidine codon is present in at least one reading frame ofa human D gene segment.
 11. The method of claim 10, wherein the readingframe is a hydrophilic frame of the human D gene segment, and thehydrophilic frame comprises a nucleotide sequence that encodes the aminoacid sequence selected from the group consisting of SEQ ID NO: 46, SEQID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56,SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO:66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ IDNO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQID NO: 86, and a combination thereof.
 12. The method of claim 1, whereinthe non-human animal is a rodent selected from a rat, a mouse and ahamster.
 13. The method of claim 1, wherein the lymphocyte is a B cell.14. A nucleic acid comprising the rearranged human immunoglobulin heavychain variable region gene sequence produced by the method of claim 1.15. The nucleic acid of claim 14, wherein the nucleic acid furthercomprises a human constant region gene sequence operably linked to therearranged human immunoglobulin heavy chain variable region genesequence.
 16. The nucleic acid of claim 15, wherein the human heavychain constant region gene sequence comprises a modification thatincreases an affinity of a C_(H)2-C_(H)3 region of an IgG heavy chainconstant region amino acid sequence to neonatal Fc receptor (FcRn) at apH ranging from about 5.5 to about 6.0, wherein the modification is amutation in the IgG heavy chain constant region amino acid sequenceselected from the group consisting of M428L, N434S, V259I, V308F, N434A,M252Y, S254T, T256E, T250Q, H433K, N434Y, and a combination thereof. 17.A cell comprising the nucleic acid of claim
 14. 18. A method ofobtaining a cell that expresses a human immunoglobulin heavy chainvariable domain with at least one histidine comprising: isolating alymphocyte from a non-human animal that comprises in its germline genomean unrearranged human immunoglobulin heavy chain variable regionnucleotide sequence that comprises an addition of at least one histidinecodon or a substitution of at least one non-histidine codon with ahistidine codon, wherein the added or substituted histidine codon is notencoded by a corresponding human germline heavy chain variable regiongene segment, and wherein the added or substituted histidine codon ispresent in a complementary determining region 3 (CDR3) encodingsequence.
 19. The method of claim 18, further comprising producing ahybridoma from the isolated lymphocyte, wherein the hybridoma expressesa human immunoglobulin heavy chain variable domain with at least onehistidine in the CDR3.
 20. The method of claim 18, wherein the non-humananimal further comprises in its germline genome an unrearranged humanimmunoglobulin light chain variable region nucleotide sequencecomprising unrearranged human V_(L) and unrearranged human J_(L) genesegments; and wherein the lymphocyte expresses a human immunoglobulinlight chain variable domain that is cognate to the human immunoglobulinheavy chain variable domain.
 21. The method of claim 18, wherein theunrearranged human immunoglobulin heavy chain variable region nucleotidesequence further comprises an addition of at least one histidine codonor a substitution of at least one non-histidine codon with a histidinecodon in a CDR1 encoding sequence, a CDR2 encoding sequence, an Nterminal encoding sequence or a loop encoding sequence
 22. The method ofclaim 18, wherein the CDR3 encoding sequence is selected from a humangermline V_(H) gene segment sequence, a human germline D gene segmentsequence, a human germline J_(H) gene segment sequence, and acombination thereof.
 23. A cell obtained according to the method ofclaim
 18. 24. An in vitro method of making a human immunoglobulin heavychain variable domain comprising: expressing in a cell the nucleic acidof claim
 14. 25. The method of claim 24, wherein the nucleic acidfurther comprises a human immunoglobulin heavy chain constant regiongene sequence operably linked to the rearranged human immunoglobulinheavy chain variable region gene sequence.
 26. The method of claim 25,wherein the human immunoglobulin heavy chain constant region genesequence comprises a modification that increases an affinity of aC_(H)2-C_(H)3 region of an IgG heavy chain constant region amino acidsequence to neonatal Fc receptor (FcRn) at a pH ranging from about 5.5to about 6.0, wherein the modification is a mutation in the IgG heavychain constant region amino acid sequence selected from the groupconsisting of M428L, N434S, V259I, V308F, N434A, M252Y, S254T, T256E,T250Q, H433K, N434Y, and a combination thereof.
 27. The method of claim24, further comprising co-expressing in the cell a nucleotide sequenceencoding a human immunoglobulin light chain variable domain.
 28. A humanimmunoglobulin heavy chain variable domain made according to the methodof claim
 24. 29. A genetically modified immunoglobulin heavy chain locusin a germline of a non-human animal comprising an unrearranged humanheavy chain variable region nucleotide sequence, wherein theunrearranged human heavy chain variable region nucleotide sequencecomprises an addition of at least one histidine codon or a substitutionof at least one endogenous non-histidine codon with a histidine codon.